The world No.2's participation at Flushing Meadows was always in doubt after he pulled out of ATP 1000 events at the Rogers Cup and Cincinnati Masters last month.
He had initially hoped to be fit for the US Open but announced on Monday that he had lost his race against time.
Nadal – winner of 14 grand slam titles – beat current world number one, Novak Djokovic, in four sets at last year's final in New York.
The Spaniard posted on his official Facebook page: "I am very sorry to announce I won't be able to play at this year's US Open, a tournament on which I've played three consecutive finals in my last participations.
"I am sure you understand that it is a very tough moment for me since it is a tournament I love and where I have great memories from fans, the night matches, so many things.
"[There is] not much more I can do right now, other than accept the situation and, as always in my case, work hard in order to be able to compete at the highest level once I am back."
Nadal also beat Djokovic in four sets to win the 2010 US Open final.
The 28-year-old's withdrawal will mean he ends the year with just one grand slam title – achieved in Paris at the French Open, a tournament he has won nine times.