The Canadian quartet, also comprising Aaron Brown, Jerome Blake and Brendon Rodney, clocked 37.50 seconds for gold.
The gold was a seventh Olympic medal for De Grasse, the 29-year-old having already one gold (200m Tokyo), two silvers (200m, Rio; 4x100m relay, Tokyo) and three bronzes (100m Rio and Tokyo; 4x100m relay Rio).
South Africa took silver in an African record of 37.57sec and Britain bronze (37.61).
"It feels pretty amazing," said De Grasse. "To be out with these guys, my brothers, I've been with them since the beginning of time, so it's amazing.
"We talked about this moment for years. It feels good to bring it to fruition. I'm super grateful."
Brown added: "Individuals didn't go our way but when we come together we're a real strong team. You can never count us out, we feel great."
Defending champions Italy, featuring Tokyo's individual 100m champion Marcell Jacobs on the second leg, finished fourth in 37.68sec.
'Just didn't happen'
The United States, missing Covid-hit 100m champion Noah Lyles, initially crossed the line in seventh in 37.89, but were later disqualified.
Christian Coleman botched the first baton handover with Kenny Bednarek, the latter coming to an almost total standstill.
Kyree King and Fred Kerley could do nothing to drag the quartet back into the running.
"It just didn't happen," said Coleman. "Maybe we could have put in some more work, it just didn't happen.
"We practised a lot. Me and Kenny have been awesome teammates a few times over the years. We felt really confident. It just didn't happen.
"We're our own biggest critic. We're disappointed because we know we had the speed to do it. It's part of the sport that you come into it with the risk reward."
Bednarek added: "At the end of the day we knew what we could do, we came out here with a mindset of no risk, no reward. It didn't happen, it is what it is."
Kerley, who won 100m bronze in Paris, said a downpour had not affected the relay.
"We train in rain, the weather doesn't affect anything," he said. "We're here to perform. We didn't get the job time. We're not disappointed, we've just got to learn from this and keep going."
It continues the sprint powerhouse's dismal Olympic run.
The last time the US men medalled was with a silver at the 2004 Athens Games.
There was a botched handoff in the heats of the 2021 Tokyo Games, while the US were disqualified at the 2016 Rio Games and failed to medal in London in 2012 whilst also coming a cropper in the semi-finals in Beijing in 2008.
US track and field legend Carl Lewis, who won two Olympic relay golds (1984, 1992) and three world relay golds (1983, 1987, 1991), took to X to voice his displeasure at the US federation's relay programme after yet another failure.
"It is time to blow up the system," said Lewis.
"This continues to be completely unacceptable. It is clear that EVERYONE at @usatf (the US track and field federation) is more concerned with relationships than winning.
"No athlete should step on the track and run another relay until this program is changed from top to bottom."
