During the final "Jeopardy!" round, leading contestant Brian Chang answered a question with an incorrect response that was a burn towards Jennings.
The category was business of travel, and the clue was, "Adjusted for inflation, the nightly rate this company put in its name in 1962 is now $51."
The correct answer was, "What is Motel 6?" but instead Chang wrote, "What is H&R Block?"
In 2004, Jennings' famous 74-game winning streak on "Jeopardy!" came to an end when he gave the wrong answer which was also H&R Block.
"I know from experience H&R Block is sometimes the right answer but not today," Jennings told Chang. "Even though you brought back some bad memories for me, you’re still going to go home with $13,201."
Jennings' winnings total ended up being over $3.3 million.
Fans of the iconic game show series reacted to the expert trolling on Twitter.
"Lol the winner on Jeopardy today instead of going for the obvious correct answer (Motel 6) decided to answer with H&R Block to roast Ken Jennings," wrote a fan.
"Brian trolling @KenJennings with his Final Jeopardy "H&R Block" answer was worth losing some money! Ken’s response was perfect. #Jeopardy," said another.
"I heartily approve of trolling Ken Jennings with H&R Block," joked someone else.
The Emmy winner served as the host for "Jeopardy!" since 1984, presiding over more than 8,000 episodes.
"Katie Couric, Aaron Rodgers, Bill Whitaker, and Mayim Bialik will each take their turn as guest hosts of Jeopardy! As a part of each guest host’s appearance, a donation will be made to a charity of their choice," the show's official Twitter account said on Wednesday.
"Alex was a legend, revered by all of us," said executive producer Mike Richards. "I want to thank our guest hosts, who will not only help all of us celebrate his great legacy, but also bring their own touch to the show."