Oasis frontman Liam Gallagher had already hyped up a lively crowd with a musical interlude before the main event - and Dubois soon proved to be a Rock 'N' Roll Star.
Despite being champion and going against tradition, he walked first to the ring to jeers as fireworks shot up above the iconic Wembley arches.
A sea of camera phones further illuminated the national stadium when fan-favourite Joshua – his eyes firmly focused on the ring – entered to a medley which started with the Godfather theme and ended with 'War' from Rocky IV.
Only four of their 49 combined wins had gone the distance, and the expected early knockdown came in the first round from underdog Dubois when he connected with a devastating overhand right in the closing seconds.
Joshua slumped to the canvas and had still not recovered in the second. Dubois did not take a backward step, stalking his opponent around the ring and finding success with his rod-like jab.
Perhaps Joshua was still haunted by those sparring stories from several years ago when Dubois reportedly rocked him.
The 2012 Olympic gold medallist was reeling in the third as his pumped-up opponent whipped in a left hook and Joshua appeared to touch the floor with his glove. It was not counted as a knockdown, but Dubois continued the assault until Joshua was floored again.
He was dropped twice more in the third, the second ruled a slip, but the writing was clearly on the wall. Unified champion Usyk and Tyson Fury, who contest a rematch in December, watched on from ringside, scarcely believing how the fight was unfolding.
The chants of "AJ, AJ" had quietened.
After a closer fourth round, Joshua, for the first time in the fight, landed a clean punch in the fifth, only for it to spur Dubois into action.
A counter right-hand, a punch that will live forever in the memory of Dubois and all those in attendance, sent Joshua down for the final time.
The former poster boy of British boxing was left scrambling across the floor, desperately trying to get up, but unable to beat the count.
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The rebuild mission since losing a second consecutive fight to Usyk in 2022 was going smoothly, fuelled by a desire to join legends such as Muhammad Ali, Evander Holyfield, Lennox Lewis and Vitali Klitschko as a three-time champion.
But Joshua came up against a fearless and younger fighter determined to prove his worth on the world stage.
"I had a fast and sharp opponent, a lot of mistakes from my end," he admitted afterwards.
The lure of the long-awaited and even longer-mooted super-fight with Fury also suffered a major blow, even if the 'Gypsy King' loses to Usyk in December's world-title rematch.
Dubois' future, in contrast, is gleaming brightly.
"I want to get to the top of this sport and reach my full potential," he said.
His father, Stan, was in his corner and just like he did against Jarrell Miller and Filip Hrgovic, Dubois Sr willed his son into action from the opening bell.
While he is no stranger to high-pressure situations and large crowds, little could have prepared Dubois for such a momentous night on the grandest stage - and four years on from a loss to another Briton in Joe Joyce, when he suffered a fractured eye socket and was called a "quitter" for taking a knee when failing to beat the count.
Yet the Greenwich fighter revelled in the spotlight again on a night which has marked a new dawn for heavyweight boxing.
"I've been on a rollercoaster run, this is my time, this is my redemption story," added Dubois.
Source: BBC