Batgirl teams up with Robin to save Gordon from Joker and Harley Quinn at Seagate Amusement Park.
- Barbara Gordon/Batgirl
- James Gordon
- Gotham City Police Department
- The Joker
- Harley Quinn
- Joker's Henchmen
- Edward Burke (Voice Only)
- Dr. Penelope Young (referenced only)
- Batman/Bruce Wayne (reference only)
- Penguin (mentioned only)
- Starro (Cameo)
- Batgir'sl Utility Belt:
Used to be a Sartorico oil rig, but was transformed into an offshore amusement park that contains a large Ferris Wheel among other rides. The island can be seen from Gotham City during the Arkham Knight Incident.
- Dual Play is featured for Batgirl & Robin as well as Joker and Harley. Hackable objects can be interacted with more ways than one, such as first luring henchmen to a machine before blowing it up and knocking out the enemies.
- Harley Quinn sports her trademark black and red ensemble outfit from Batman: The Animated Series.
- At one point, Joker refers to Batgirl as "Bat-fake", the same insult he later referred to Terry McGinnis in Batman Beyond: Return of the Joker. Coincidentally, both characters also effectively replace Batman at those times (although Batgirl's case was more temporary than permanent).
- Joker once again used the alias of "Jack White" and manipulates someone.
- Penelope Young is referenced in an audio file.
- The Laughing Fish makes a startling cameo in the game. An animatronic of a jokerized fish appears and frightens Batgirl as she explores the Ghost Ship.
- Starro makes a cameo in a secret room between the Ferris Wheel and the Great White. In addition, a billboard features an advertisement for the Starfish Freak, which alludes to Starro's ability to take over people's minds via spores.
- The name of the character, Edward Burke, who built Seagate Amusement Park, might be a reference to the DC Comic book super-villain, Planet Master.
- Near the Great White, there is a large floating balloon of a clown with a long nose, which alludes to one of the Joker's float balloons from Tim Burton's Batman.
- Some of Joker's Henchmen, when preparing to "feed" a GCPD officer to the Kraken, comment about how they could probably revive the Kraken by extracting DNA from "orange glass" (with the GCPD correcting them by stating that it's amber and that it's actually hardened plant sap rather than glass), which alludes to the basic premise of the Jurassic Park franchise.