Florida State guard Michael Snaer hit a three at the buzzer to defeat Duke at Cameron Indoor, 76-73.
The Seminoles trailed by as many as eight in the second half, before they handed Duke its first ACC loss.
The loss ended the Blue Devils' 45-game home winning streak. It also ends their streak of 65 home wins against unranked opponents, which dates back to February 2007.
Florida State was actually the last unranked team to defeat the Blue Devils at Cameron.
After Duke's Ryan Kelly and Florida State's Xavier Gibson each made one of two free throws, Duke had a chance to tie it. Austin Rivers made a layup with under five seconds left, and the game seemed destined for overtime.
Then, Luke Loucks sprinted up the floor before dishing to Snaer for the winner.
While the announcers and Seminoles bench go crazy, Cameron falls silent. The Seminoles had won just their second game in Durham against Mike Krzyzewski.
Gibson led the way for the Seminoles with 16 points. Meanwhile, Rivers led all scorers with 19.
Duke was able to grab 12 offensive rebounds, but it wasn't enough to beat the upstart Seminoles.
Florida State (13-6, 4-1) heads to Wake Forest on Wednesday. On the same night, Duke (16-3, 4-1) will take on rival Maryland.
Florida State's win puts them at the top of the ACC, proving that the conference is wide open this year.