The system is used to warn the public about floods, tornadoes and other emergencies.
Robert Ratcliffe, the acting chief of the FCC Enforcement Bureau, said the agency "will not tolerate" misuse of the system.
“It is inexcusable to trivialize the sounds specifically used to notify viewers of the dangers of an incoming tornado or to alert them to be on the lookout for a kidnapped child, merely to advertise a talk show or a clothing store," he said in a statement late Tuesday.
"This activity not only undermines the very purpose of a unique set of emergency alert signals, but is a clear violation of the law."
The TBS commercial used emergency alert tones to promote the appearance of Jack Black on "Conan" on April 26, 2012, according to the FCC.
TBS can still challenge the fine, which has not been finalized.
The cable station did not respond to a request for comment Wednesday, but in documents filed with the FCC, it said the promotion was produced in a tight timeframe, and the production team did not submit it for review.
The company said that since May 2012, all similar promotions for O'Brien's show have gone through a standards and practices review.
The FCC said there has been a spike in complaints about misuse of emergency alert tones.
The agency also reached an agreement with a Kentucky Television station, WNKY, for simulating the emergency alert sound in an ad for the "The Fan Wear & More Store."
The local station agreed to pay $39,000 and implement a compliance plan to prevent future violations.