The top seed had been hoping to win a second successive Major crown after emerging victorious at Wimbledon in July but she slipped to a 6-2 7-6 to the Czech, who reaches her first Grand Slam final.
As a result of the defeat, Williams will lose her standing as world number one after 186 consecutive weeks at the top of the WTA rankings.
The opening set lasted just 26 minutes as Pliskova broke Williams's serve on three occasions, but it was a tighter affair in the second as Williams responded.
The American revealed after the match that she had been hampered with a sore knee throughout the tournament but regardless of the injury, Pliskova continued to play the better tennis on the Arthur Ashe Stadium court.
After a break apiece, the set went to a tie-break and it was Pliskova who prevailed after Williams double-faulted on match point.