An Easter egg/reference relating to The Scarecrow can be found on a barge in the docks area. When on the barge, use the sequencer gadget to solve the code "City of Terror". A panel opens. Once inside, you are in a room filled with neon bug zappers. At the end of the hall, there is an unconscious inmate sitting in a chair. Upon zooming in on him, he is startled awake and gives a loud shriek. He goes back to sleep. It is assumed that he is a victim of The Scarecrow's experiments.
It is heavily implied at one point in the game that Harley Quinn is or was pregnant. This is suggested by examining Harley's costume on a dummy and panning down to the floor. At the dummy's feet is a box reading "Fruitful Endeavors pregnancy test". Lying beside it is a positive test.
When you go to Two Face's courthouse and you enter the back way; you can go down to the cell next to the Calendar Man's cell. When you're in that cell turn on detective mode, look up (make sure you are under them), and punch. When you do that they somehow notice you and run through the door and come to fight you.
There are several references to Lost (2004), the ABC television show. One when flying around Arkham City, an inmate asks another, "So, did they ever explain what the island was?" And later, more dialogue: "...why did it end in a church?" Writer Paul Dini was part of the "Lost" writing staff for the first two seasons before leaving the series.
This is the first known project where Tara Strong has voiced the role of Harley Quinn, which has predominantly been voiced by creator Paul Dini's friend Arleen Sorkin. Strong is better known in the Batman animated universe in the role of Barbara Gordon, aka Batgirl and in this game aka Oracle.
Peter MacNicol voices The Madhatter - a villain obsessed with collecting Batman's cowl. MacNicol also voices X The Eliminator - a villain similarly obsessed with collecting part of the hero's outfit (his crest) - In Harvey Birdman Attorney At Law.