Djokovic showed his frustration after losing serve to trail 6-5 against Spain’s Pablo Carreno Busta.
The Serbian world number one, 33, took a ball out of his pocket and hit it behind him, striking the female line judge in her throat.
After a lengthy discussion, he was defaulted by tournament officials.
A United States Tennis Association statement said: “In accordance with the Grand Slam rulebook, following his actions of intentionally hitting a ball dangerously or recklessly within the court or hitting a ball with negligent disregard of the consequences, the US Open tournament referee defaulted Novak Djokovic from the 2020 US Open.
“Because he was defaulted, Djokovic will lose all ranking points earned at the US Open and will be fined the prize money won at the tournament in addition to any or all fines levied with respect to the offending incident.”
Djokovic was the heavy favourite to win the men’s singles title at the US Open, which is being played behind closed doors and is the first Grand Slam to take place since the beginning of the coronavirus pandemic.
Going into the encounter with 20th seed Carreno Busta, Djokovic had not lost a singles match in 2020.
He was aiming for an 18th Grand Slam triumph to move closer to rivals Rafael Nadal and Roger Federer, who are not playing in New York, in the race to finish with the most men’s major titles of all time.
Djokovic had been playing well up until the game where he lost serve and had three set points at 5-4 before Carreno Busta fought back from 0-40 down.
However, the world number one also showed a flash of his temper during that game by whacking a ball into a sideboard after the Spaniard brought it back to deuce.
Leon Smith, Great Britain’s Davis Cup captain, said he was “surprised” Djokovic did not get a warning for that incident, and felt one might have prevented the controversy which followed.
After Djokovic was defaulted, Smith added: “There has to be consistency, if someone hits a ball with that much venom and temper, call a warning.
“The second one can be dangerous and so it proved to be.”
In what proved the final game, Djokovic fell and hurt his shoulder, immediately calling for a medical timeout when trailing 0-30. Following treatment, the match resumed with Carreno Busta sealing the set three points later.
It was then that Djokovic hit the ball away, striking the line judge.
Djokovic appeared to plead his case to tournament referee Soeren Friemel and Grand Slam supervisor Andreas Egli during a long conversation at the net.
Eventually, however, he accepted his fate and shook hands with Carreno Busta, who looked shocked by what had happened as he waited for a decision in his chair.
Djokovic left Flushing Meadows without doing his news conference.
His defeat means there will be a new male Grand Slam champion for the first time since Marin Cilic won in New York in 2014.
