During a battle with Jasper, the Crystal Gems, Steven Universe, and their archenemy Peridot with a good number of Flask Robonoids have been transported to Gotham City. While the Flask Robonoids attempt to steal various components from labs all around Gotham to work out a means of returning home, as well as abducting a scientist doing research into cross-dimensional travel, the Crystal Gems intercept the various thefts, although the Flask Robonoids still depart with their gathered equipment.
While Batman is puzzled at the fact that the crimes were committed by gems with a specific focus on a key area of fighting skills - dismissing the League of Assassins as candidates, as their members have a broader range of training and Ra's al Ghul would have alerted Batman to his presence by now. By now the Crystal Gems have set up a new base in the sewers, but Killer Croc stumbles upon their lair while tracking Batman. After defeating his men and then Croc himself, the Gems are forced to flee due to Croc knowing the location of their new base in the sewers. The Crystal Gems retreat to the surface in time to discover the Batmobile, just as Batman departs the latest theft, having been confronted by Peridot and learning that the Flask Robonoids and the Crystal Gems are actually adversaries rather than allies. However, when he returns to his car, he finds the Gems milling about it.
After a brief fight between the Crystal Gems and Batman (where Batman easily beats all of the Gems at once), the Crystal Gems escape when Steven intervenes and distracts Batman with a dance. The Crystal Gems research Batman at an internet café, and most of them come to admire him, while Garnet just believes him to be a distraction.
Having recovered Garnet's shades during the fight, Batman takes it to Lucius Fox as Bruce Wayne. Fox's analysis confirms that the eyewear is from another universe, made of a slightly different version of the glass they are familiar with, but it is reverting to this reality's 'local' version of glass. He also notes that there is a strange evolution pattern in a blood sample that Bruce provided him with, but the evolution pattern is incompatible with their laws of physics and anything evolved with it will thus revert to its non-evolved state. Unbeknownst to Batman, Steven followed him into the Batcave and learned of the decaying gem serum.
Learning about a stolen power generator that could allow them to power the equipment they need to return home, Peridot and the Flask Robonoids make a 'deal' with the Penguin to acquire it, only for the Flask Robonoids to kill all of Penguin's men. However, the Penguin convinces Peridot to spare him by arguing that he can provide useful local knowledge.
Reuniting with the Crystal Gems and explaining the situation, Steven then leads the Gems to the Batcave to ask for help. Although Batman is initially hostile, Steven catches Batman off guard by calling him by his real name, which helps them form an uneasy peace. Having explained their situation to Batman, Batman, Steven Universe and the Crystal Gems lead an assault on the Iceberg Lounge, where Peridot intends to activate a portal that will return him to her world, also planning to create a permanent link between their worlds so that she can conquer Beach City and Gotham. However, when the Gems and Batman attack, Peridot destroys the portal and kills the scientist who helped her build it, gloating that she can rule in this world while the 'clods' and Steven will revert to normal gems to use for her jewelry. Pearl engages Peridot in battle, but Peridot loads a stolen vial of the decaying gem serum into her laser gauntlet and fires at Pearl's chest and incapacitates her. As she escapes, Peridot discovers that her robots have been killed by the League of Assassins, and she is greeted by Ra's al Ghul, who offers her an alliance.
With Peridot unaccounted for, the Gems remain in the Batcave and Wayne Manor, with Garnet brainstorming possible alternatives to the evolution decay with Cyborg, while Steven trains against Batman and Amethyst enjoys herself. Eventually, Pearl snaps at the lack of progress made in finding a cure, accusing Batman of being simply "a rich boy playing at being a hero" with no idea about true loss and sacrifice, storming off in a huff.
In another part of Gotham, Peridot and Ra's have assembled another portal to intercept an inter-dimensional signal Peridot detected when creating the original device; the signal is revealed to be Lapis. Lapis easily beats the Flask Robonoids and League of Assassins before facing Peridot and Ra's.
While Pearl is walking through Gotham in disguise, Batman pulls up beside her in the Batmobile and takes her to Crime Alley, where he tells Pearl about the death of his parents and how that loss drives him to become Batman. As Pearl considers this and finally starts to accept Batman, the other Crystal Gems alert her to an energy surge in Gotham that they identified as another portal. The others took (and crashed) a second Batmobile to the source of the signal. Rescuing Lapis from the Flask Robonoids, the Gems are shocked to learn that Batman's rogues gallery have stolen four full vials of gem serum that Lapis had brought to stabilise Pearl's condition and the other three for the other Gems just in case... and, before Lapis lost consciousness, she overheard the villains say that they intend to use the gem serum in "Arkham".
Responding to the Batsignal, Batman, Garnet and Pearl meet with Commissioner Gordon at the roof of the GCPD and learn that the Penguin has turned informer, revealing that Peridot and Ra's are intending to attack Arkham. As Pearl collapses due to the decaying gem serum in her system, Damian returns to the Batcave to find Steven Universe and Amethyst playing video games on the Batcomputer and flips over Lapis when she underestimated him, resulting in a brief fight between the Gems versus Damian until Batman returns to confirm that the Crystal Gems are his allies. As Damian reports that the League of Assassins are coming to Gotham en masse, Lapis reveals that she was provided with equipment that would allow the Gems to return to their dimension, and Batman tells the Crystal Gems to get home before Pearl's condition decays any further. Despite Steven warning Batman not to underestimate Peridot, Batman and Damian head to Arkham, confident that they can beat Peridot, only to be confronted by the Penguin, who had been caught by Peridot and mutated into a black spinal Crystal Gem. He is backed by a mass of gem-contaminated inmates ( Mr. Freeze as opaque, Bane as an ruby (he is still male), The Riddler as an emerald, Two-Face as a sunstone, Scarecrow as a citrine, etc.), who move in for the attack.
Batman and Robin are able to escape Bane and knock Harley Quinn unconscious, before Batman is trapped by the mutated Mr. Freeze and Poison Ivy. Confronted by al Ghul and Peridot, he learns that the two arch-villains intend to use the gem serum in every major city of the world, throwing human civilization in chaos and thus paving their ascent to total world domination. Seeing all of this in the Batcave, the Crystal Gems decide to stay and risk their lives to save Batman, despite being given the chance to escape back to their own dimension with Lapis. The Gems arrive just as soon as The Joker (now mutated into a sugilite and is still male) is about to kill Batman, and Pearl knocks him off while Garnet gets Batman. While Peridot fights off the remaining inmates, the Dark Knight battles Peridot inside his latest Gem combat armor, which was given Gem themed upgrades by Garnet, while the Crystal Gems fight Ra's al Ghul. Although Peridot severely injures Batman and breaks most of the Gem suit, Batman manages to knock his opponent to the ground, and Peridot (who had defeated all the inmates), finishes her off. With the Gems beating her down, and the League of Assassins disarmed and captured by Damian and the GCPD, al Ghul escapes with a smoke bomb.
Just as the Crystal Gems wonder how they will get back home, a dimensional portal opens and disgorges Connie and Lars, after Connie and Lars managed to pinpoint their frequency after Lapis had returned alone. The Gems round up the Flask Robonoid clan and Peridot and send them back to their original dimension and Batman gives the last vial of gem serum to Pearl. Before they leave, Garnet gives Batman her shades out of respect and tells him that he's got a good family and has another if he ever shows up in their dimension. Batman and Robin return to the Batcave where they learn from Commissioner Gordon that the situation at Arkham is being stabilized until the gem serum has decayed naturally, turning the inmates back to normal within a week. Damian remembers that it's the anniversary of the death of Bruce's parents and volunteers to leave, but Bruce insists that he stays and they decide to work on the Gem Suit together.