The Czech 10th seed crushed the 34-year-old's hopes of moving ahead of Steffi Graf's Open-era record 22 major triumphs with a 6-2 7-6 (7-5) triumph to reach her first major final.
Highlights: Pliskova stuns Serena
It marked the second consecutive year Williams lost in the last four at Flushing Meadows while chasing history. She came into last year's semi-finals as the three-time defending champion and two wins shy of a rare calendar-year Grand Slam but was upset by Roberta Vinci.
Pliskova, the first Czech woman since Jana Novotná in 1998 to reach the semi-finals at Flushing Meadows, showed no early signs of nerves and surprisingly broke in just the third game after the world No.1 misfired her forehand beyond the baseline.
Mouratoglou: Serena couldn't cope with pain in her knee
The 6ft 1in Czech pummeled a series of aces to consolidate the break while the errors continued to fly off Williams' racket.
Despite making two double faults in the sixth game Pliskova held on to her serve to stay ahead and then broke the six-time US Open champion again to move a double break ahead. And she completed an emphatic opening set in just 26 minutes to stunned spectators on Arthur Ashe Stadium.
When Serena won her first US Open title in 1999, Pliskova was just seven-years-old, and it was her younger opponent who was staying calm under the lights to maintain her focus against a more aggressive Williams at the start of the second set.
Pliskova: I can't describe what it's like... it's a dream
The world No. 11 had never gone past the third round of a major tournament before but arrived in New York on a high, having just beaten Angelique Kerber to claim the title in Cincinnati last month while Williams was playing in her 11th semi-final at Flushing Meadows.
She knocked out Venus Williams in the last 16 and now had her younger sister in her sights after forcing further errors from the American to move 3-2 ahead.
Amazing recovery from Serena sees her win point off Pliskova
Pliskova was unable to take advantage though as Williams earned her first break points before taking advantage to restore parity.
The set went to a tiebreak and it was Pliskova who won through to the final after Williams double faulted and lose her staus as world No.1 to Angelique Kerber.
"I don't believe it. Actually, I do believe it," Pliskova said in a courtside interview after the game. "I always knew I have a chance to beat anybody if I'm playing my game.
"But this is something amazing.
"Serena, she's a champion so it's never easy to play her. You saw even when she was down a break she's still fighting so it's very hard."
Williams however refused to use fatigue as an excuse for her defeat.
"OK, I'm not going to repeat myself. I wasn't tired from yesterday's match," said Williams. "If I can't turn around after 24 hours and play again then I shouldn't be on tour.
"I have been having some serious left knee problems. I wasn't tired. Fatigue had absolutely nothing to do with it.
"I wasn't able to move the way I wanted to move."