Williams, a six-times winner at Flushing Meadows, has had an injury-marred season and limped out of her first-round match at Wimbledon in tears due to the leg injury.
The 39-year-old American, who has been on the hunt for an elusive 24th major, skipped the Olympics in Tokyo and also opted out of the Cincinnati Masters, a tune-up event to the US Open.
"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," Williams wrote in a statement on Instagram on Wednesday.
"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 everyone from afar."
Williams is the latest big name to pull out of the tournament after reigning men's champion Dominic Thiem and four-times winner Rafa Nadal ended their 2021 season due to injuries.
Her withdrawal from the tournament casts further doubt over whether she can match Margaret Court's record of 24 major titles, as she turns 40 next month.
Williams has reached the finals in four Grand Slams since giving birth to daughter Olympia four years ago but has not triumphed in a major since picking up her 23rd title at the 2017 Australian Open, losing in straight sets on each occasion.
Speaking to reporters on Tuesday, ESPN analyst and Hall of Famer Pam Shriver said she was uncertain whether Williams could break through, given the depth of talent in women's tennis.
"Unfortunately time is really running out," said Shriver.
"Since she came back from maternity leave she did get to four major finals, unfortunately was never in one of them really... She certainly had opportunities."
The main draw of the US Open gets under way in New York on Monday.
