Chief Miles O'Hara first appeared in the Batman episode "Hi Diddle Riddle" (January 1966). He was first mentioned in comics in World's Finest #159 (August 1966) and first appeared on-panel in Detective Comics #461 (July 1976), though he was obscured by an object. He briefly replaced Jim Gordon as commissioner beginning in Wonder Woman #281 (July 1981).
His first Post-Crisis appearance was as Police Chief Clancy O'Hara in Batman: Dark Victory #1 (December 1999), where he was the first victim of the Hangman.
King Tut now has a character spotlight!
Take a look at Egghead's Character Spotlight!
Bookworm and Marsha, Queen of Diamonds
Although these villains from the Batman tv series (1966-1968) haven't yet made a real appearance in comics, they were mentioned by the Riddler in Secret Origins Special #1 (June 1989). Unfortunately, until the rights to the 60s series get sorted out, they won't be making any actual appearances any time soon.
UPDATE: They have gotten sorted out, so take a look at Bookworm's Character Spotlight!
Batgirl (Barbara Gordon)
Batgirl is an interesting case. She first appeared in Detective Comics #359 (January 1967) and did not appear in Batman until the episode "Enter Batgirl, Exit Penguin" (September 1967), so she technically should not be on this site. But the only reason she exists is because the producers wanted more female characters to put on the show, so I've included her here. (A similar, but ultimately different, case can be found in Renee Montoya.)
Aunt Harriet's Last Name and Widow Status
Despite common misconception, Aunt Harriet did appear in the comics before the tv show, making her first appearance in Detective Comics #328 (June 1964). But that's all she was: Aunt Harriet. It wasn't until the first episode of the tv show (January 1966) that she was Aunt Harriet Cooper, widow of the late Mr. Cooper. These facts were brought over to the comics in Detective Comics #373 (March 1968), which incidentally brought over the name "Mr. Freeze" from the show as well.
The Batmobile's afterburner
The Batmobile's famous afterburner first appeared in the Batman tv series (1966). It first appeared in comics later that year in an issue of The Brave and the Bold.
(Note: Mr. Freeze, The Riddler, and the Batmobile will be featured in a later post.)