A seventh US Open title would see the American equal Margaret Court's Grand Slam haul but Williams has lost her last two semi-finals in NY.
Williams, seeded 17th, trailed in the first set but hit back to win 6-4 6-3 against the Czech eighth seed.
She will play No 19 seed Anastasija Sevastova in the semi-final on Thursday, after winning her 14th consecutive quarter-final in a Major at the Arthur Ashe Stadium.
The Latvian made her third consecutive trip to the NY quarter-finals this year and knocked defending champion Sloane Stephens out to reach her first Grand Slam semi-final.
Williams had to fight off three break points against her in the fifth game and was unable to convert two break points on Pliskova's serve in the sixth game.
Sevastova advances to a Slam semi-final for the first time in her career, having reached three consecutive quarter-finals at the US Open, and becomes the first Latvian woman to reach the semi-finals in NY.
"It's cruel, sometimes, tennis", Thiem said, calling the loss "the first really epic match I've played".
The six-time U.S. Open champion is looking for her 24th Slam singles title and seventh U.S. Open.
It took five deuces for Williams to finally hold her serve, it was clear at this point that advantage was in the Czech's favour with Williams having to work a lot harder.
This time a year ago, Serena Williams was battling through a very different challenge as she gave birth to her first child, Alexis Olympia.
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Romelu Lukaku opened the scoring for the Red Devils as he latched onto a brilliant cross from Alexis Sanchez . Writing on Twitter, Rashford said: "Emotions got the better of me, I shouldn't of reacted like that.
"I'd been a couple of steps away at the last Grand Slam, so I'm definitely not ahead of myself", said Williams, who fell to Angelique Kerber in the Wimbledon final in July. "I said, 'I got to try even harder'".
Williams has done her part, making her way to the last four with her usual efficiency while throwing in an occasional wobble just to keep things interesting but not scary. She then served her way through a love game to claw back to 4-2.
Serena Williams is walking down the tunnel now and stops for her pre-match interview with ESPN's Tom Rinaldi. He's into the semifinals for the second year in a row, where he lost to top-ranked Rafael Nadal last year.
Williams is now a potential contender for winning the US Open this year. You can't control the weather, can't control what the tournament is going to do. He was knocked out before the quarterfinals of a major event for the first time since he lost in the third round of the 2015 Australian Open.
The American was rattled, and it showed, as she was broken again at the start of the second set.
"When you don't play the big points well and don't take your opportunities early, that could affect the outcome of the match", Stephens said.
"He's a close friend on tour, a great guy and I wish him the very best. he is young and has plenty of time to win tournaments".
Indeed Stephens fought back to narrow the gap to 4-3, but Sevastova grabbed another break with a well-timed drop shot for a 5-3 lead and sealed the victory on her third match point when a tired Sephens put a backhand into the net.
"She's playing with the same power. I don't think there's any change with her game", Pliskova said.