In a post on social media, the 39-year-old American great said she needed time to allow her body to "heal completely" from a torn hamstring.
Williams joins defending men's champion Dominic Thiem, plus Roger Federer and Rafael Nadal, in pulling out of this year's final Grand Slam.
The tournament, at Flushing Meadows in New York, begins on 30 August.
Williams wrote on Instagram: "After careful consideration and following the advice of my doctors and medical team, I have decided to withdraw from the US Open to allow my body to heal completely from a torn hamstring.
"New York is one of the most exciting cities in the world and one of my favourite places to play - I'll miss seeing the fans but will be cheering on from afar.
"Thank you for your continued love and support. I'll see you soon."
Williams suffered the injury to her hamstring during her first-round exit at Wimbledon in June, when the seven-time champion was forced to retire against Belarusian Aliaksandra Sasnovich.
She pulled out of last week's Western and Southern Open in Cincinnati in order to focus on her rehabilitation.