The injury, sustained in his fourth-round win over Francisco Cerundolo, ended the Serb's quest to win a record-extending 25th Grand Slam trophy and will result in him losing his top ranking to Italian Jannik Sinner later this month.
"Due to a torn medial meniscus in his right knee (discovered during an MRI scan), Djokovic, who was supposed to play Casper Ruud in the quarter-finals tomorrow, has been forced to withdraw from the Roland Garros tournament," tournament organisers said.
Last year's runner-up and seventh seed Ruud will now advance to the semi-finals, where he could take on fourth seed Alexander Zverev or 11th seed Alex de Minaur.
Djokovic said on Instagram that he was disappointed to pull out of the tournament that he has won three times.
"I played with my heart and gave my all in yesterday's match," Djokovic said. "Unfortunately... I had to make a tough decision after careful consideration and consultation.
"I wish the best of luck to the players competing this week and sincerely thank the incredible fans for all of the love and continued support."
Sinner said it was not the way he hoped to take the top spot, adding Djokovic's exit was a loss for the tournament.
"It's not the way we all were expecting, actually. He had two very long matches, tough matches, five sets, so it's tough. The first one he finished really late also," Sinner said.
"It's tough also for the tournament. Novak retiring, it's always tough."
Monday's marathon match was the second straight clash in which Djokovic was taken to five sets following his epic against Lorenzo Musetti and he has spent over nine hours on court in the last two rounds at the year's second major.
The Serb said he had been carrying an injury for a couple of weeks and that the problem had flared up when he slipped during his match against Cerundolo, which he completed with the help of pain-killers.
The injury could also cast doubts over his fitness ahead of next month's Wimbledon and the Paris Olympic Games that follow, although Ruud was optimistic as he wished Djokovic well on X.
"Speedy recovery Nole. Hope to see you back and healthy on the grass," Ruud said.
It has been a season to forget for Djokovic after his bid for a record-extending 11th Australian Open title was ended by eventual champion Sinner in the semi-finals, before he lost to lucky loser Luca Nardi early at Indian Wells.
Having been stunned by Ruud in the Monte Carlo semi-finals, Djokovic was thrashed 6-2 6-3 by Alejandro Tabilo in the third round in Rome last month, two days after being hit on the head by a fan's water bottle while signing autographs.
He was also beaten in three sets by unheralded Tomas Machac in the Geneva semi-finals ahead of Roland Garros.
Djokovic is still without a trophy this year after one of the most dominant campaigns of his glittering career in 2023, during which he claimed three of the four Grand Slam titles.
