When you lose to Bane, one of the game over sequences shows Bane breaking Batman's back, which is a direct reference to the "Knightfall" comic storyline in which Bane first appeared.
Entered into the Guinness Book of World Records for most critically acclaimed superhero video game of all time.
In one of the Scarecrow audio tape interviews, you will hear mentions of Dr. Murphy and Dr. Combs. Cillian Murphy plays Scarecrow in Christopher Nolan's Batman film series and Jeffrey Combs voiced Scarecrow on the later episodes of The New Batman Adventures (1997) and in the video game Batman: Rise of Sin Tzu (2003).
There is a hidden room in the Arkham mansion, where after applying three sprays of the explosive gel on a wall, players can enter and see blue prints, pictures and plans for the sequel, Batman: Arkham City (2011). Using detective vision won't give hints as to where the room is. It was also so well hidden that Rocksteady didn't say anything about it until 6 months after the game's release.
The sound of the explosive gel gadget when you use it was actually taken from a whipped cream can being used.