Two-Face is never featured in this show, nor is his alter ego, Harvey Dent due to an agreement with Warner Brothers not to use him because of his planned use Batman Begins (2005) and The Dark Knight (2008). A new character named Ethan Bennett was created in his place and featured many elements from Harvey Dent. Bennett also had a tragic accident that caused him to become a villain.
For Robin's costume, Jeff Matsuda said he used the Tim Drake Robin costume from the comics as a starting point and took out what he didn't like. Some of the changes were bringing back the traditional pixie shoes, making the "R" symbol a yellow circle with a black "R" (like it was in The New Adventures of Batman (1977)), and giving Robin a modern version of the Dick Sprang styled hair from the comics.
This is the first Batman cartoon to introduce Batgirl before Robin. Batgirl was introduced before Robin simply because Robin still being used on Teen Titans (2003), which had been renewed for a 5th season, which aired on a different network. Robin in this show looks exactly he did on Teen Titans (2003) albeit a few years younger. He also uses Bird-A-Rangs and uses a bow staff as a weapon just as he did on Teen Titans (2003).
Batman's cowl featured shorter ears, because it was more boxer like according to Jeff Matsuda, and the nose is flatter than previous versions. In Season 4 The Batman's was chin was redesigned to be more square and less pointy.