Her opponent in the final will be new world No 1 Angelique Kerber, who reached her third major final this year after dispatching Caroline Wozniacki in straight sets.
In the second semifinal, Kerber dispatched Wozniacki in relatively straightforward fashion, earning two breaks in the first set and getting another in the first game of the second set.
The disappointment is doubled for Williams as it means she will also surrender the world number one ranking at the end of the tournament to Germany's Angelique Kerber.
The sore right shoulder that developed in the days after the American won Wimbledon to tie Steffi Graf's Open-era mark of 22 major championships does not appear to be a problem any long. Though the German lost, she was confident that she would get another chance to prove she is the world number one.
Serena Williams entered Thursday evening's semifinal match at the US Open in Queens, New York, as the overall top seed in the women's tournament.
With a third Grand Slam final of the year on the horizon for the German, who finished last year ranked 10th, couldn't be distracted despite realising her dream rise to the top of the world rankings.
The opening set lasted just 26 minutes as Pliskova broke the Williams's serve on three occasions but it was a tighter affair in the second as Williams responded. Pliskova is only the fourth woman to beat both Williams siblings during the same Grand Slam tournament. She knocked out from US Open women's semifinals on Thursday.
The temperature was in the low 80s, and the air was muggy and still, and Williams kept using the pleats of her black-and-pink skirt to wipe her sweaty palms between points.
She flubbed shot after shot, 31 unforced errors in all.
The loss was devastating for Williams, who was also seeking to break Chris Evert's record of six U.S. Open titles.
But she believes she laid the foundations for her shock semi-final victory by saving a match point in her gruelling three-set win over Venus in the fourth round.
She also said she was unaware that Williams was carrying a knee injury. "There was no match".
Pliskova was in no mood for sympathy on and off the court though as she ruthlessly conquered the American 6-2 7-6 (7-5). She averaged 109 miles per hour on her first serves Thursday - faster than the younger Williams, known for the best serve in the women's game - and leads the tour in aces this year.
Williams added she is used to playing matches on back-to-back days given it is commonplace at most events outside the grand slams.
"Then I stepped into the ball a bit more then and went for my shots and started going in and I started finding a rhythm".
According to Mouratoglou, Williams received treatment all day and scrapped most of her warm-up. "I was trying not to think about it too much the last few weeks". She gets good angles.
The odds were incredibly lopsided for a semifinal match of a Grad Slam, regardless of the fact that perhaps the best female player of all-time was playing. Pliskova sat in her changeover seat, leaned back and exhaled.
Into the tiebreaker they went, and Pliskova jumped ahead 3-0.