Near the end of the game, Hugo Strange says "This is just the beginning", a line said near the end of Attack of the Clones by Count Dooku, who Corey Burton has played.
A subtle one: Joker attempts to force Mr. Freeze to develop a cure for him by holding his wife hostage. Joker is voiced by Mark Hamill, whose first voice in Batman: The Animated Series was Ferris Boyle, the guy responsible for Mr. Freeze, and Mark's performance as him also landed him with his voicing The Joker in that series. Furthermore, by reading one of the riddles called "Who can BOYLE and Freeze at the same time?" and listening to the Mr. Freeze audio tapes, you find out that Ferris Boyle is canon in the Arkhamverse.
MPT Anne Bishop is voiced by Kimberly Brooks. Given the character's looks and personality (her first question when Batman rescues her is where's Riddler, clearly intending to remove his spine), chances are that Ashley Williams will come to mind.
Beam Me Up, Scotty!: Many have misquoted or misinterpreted Mark Hamill as saying this game will be the last time he plays The Joker. He clarified that what he meant was he wouldn't continue playing The Joker if he felt that he couldn't top his performance, he wanted to leave on a high note. That said, this would seem to suggest that he has no plans to play the Joker again in the future. Then came Batman: Arkham Knight...
Blooper: As the generator slams Clayface into the Lazarus Pit and explodes, the vial holding the cure is in Batman's left hand, but when he comes to, it's in his right one. Until Arkham Knight confirmed the Joker died, some people speculated that it was intentional, and the Joker took some of the cure and was faking his death. After Knight, though, it's become clear that it was indeed a screw-up.
Development Gag: At one point, Batman overhears thugs talking about the rumor that Harley Quinn used to be a man. Harley Quinn was actually motion-captured by a man, and they even released a fake reveal trailer to tease fans about it.
Fan Nickname: Robin's is RobinHood.* In his very first appearance, Dick Grayson said his name was "Robin, as in Robin Hood... not robbing hoods!"... hey, it was 1940.
The Other Darrin: Tara Strong replaces Arleen Sorkin as Harley Quinn, David Kaye replaces Tom Kane as Commissioner Gordon, and Grey DeLisle replaces Tasia Valenza as Martha Wayne. The latter two are perplexing since, as noted above, Tom and Tasia returned from the first game to reprise the respective roles of Quincy Sharp and Poison Ivy.
In an interview, director Sefton Hill said they wanted to include a reference to the infamous Bat-Shark Repellent Spray from the Adam West seriesnote Reportedly, when confronted by "Tiny" in the Iceberg Lounge, Batman would break out the repellent... then open it up to reveal a set of brass knuckles, but ended up nixing it because they felt it was a Level Breaker. There's still a subtle reference; if you get killed by the shark, one of the possible hints reminds you to remember Batman's shark repellent: a flurry of blows to drive it off. In one room, you can listen to the Riddler mutter about ways to take down Batman, and ponders taking Penguin's shark, only to say that there are "too many variables", and that Batman likely has some sort of "shark contingency".
The plot itself borrows ideas from Knightfall and No Man's Land, among other famous Batman stories. A lot of the Joker/Batman dynamic is reminiscent of The Killing Joke.
Arkham City was released the same day as the Animated Adaptation of Batman: Year One and shares a lot of the same voice talent: Grey DeLisle (Catwoman, Vicki Vale, Stacy Baker, Martha Wayne) is Barbara Eileen-Gordon and Vicki Vale, Steve Blum (Killer Croc and Sickle) is Stan, Fred Tatasciore (Bane, Solomon Grundy and Mr. Hammer) is Arnold Flass and Johnny Vitti, and Danny Jacobs (Victor Zsasz) is Flass's lawyer.