The loss sees Williams relinquish the world No.1 ranking to German Angelique Kerber.
Pliskova broke the Willaims serve twice in the openign set, as the now former world No.1 served six double faults for the match, including on match point to hand Pliskova victory, in an uncharacteristically error riddled match.
Pliskova advanced to her first Grand Slam final, with he previous best result at a slam the third round.
She is the first Czech player to reach the US Open final since 1993 and she becomes just the eighth player in history to defeat both Williams sisters in the same tournament.
The 24-year-old also recorded her sixth win against top-10 opponents this season.
She will face either Caroline Wozniacki or Angelique Kerber in the final.