With Usyk set to fight Tyson Fury in a rematch in December, the Ukrainian was forced to vacate IBF belt as a result, just five weeks after becoming the undisputed heavyweight champion with a split-decision victory.
Usyk's mandatory IBF title defence against challenger Filip Hrgovic was left pending and Dubois became the new interim IBF heavyweight champion by beating the Croatian earlier this month.
"I'm aiming to be the best. AJ (Joshua) has been the King for a long time but on the night I need to be the 'King slayer'. That's the goal, the mission I have," Dubois said at the news conference.
"I'm ready to let my fists do the talking. I'm 100% ready to go and to train like a beast."
Dubois has a 21-2 record with 20 knockouts while Joshua's record is 28-3, including 25 knockouts.
Usyk beat two-time heavyweight champion Joshua twice - first in 2021 to seize the WBA, WBO, IBF and IBO belts and again in 2022 when he defended the belts - but the Briton has won his last four fights.
"I went out to watch his last fight... Dubois has been on my mind for a while and he will be for the next 12 weeks," Joshua said.
"Whoever was going to be there, I was ready to fight in September. It just happened to be him."
When questioned about the age difference, Joshua said: "It don't matter, we're in peak condition. Age is just a number."
Usyk had knocked out Dubois in nine rounds last year in a bout overshadowed by a low blow controversy when the Ukrainian went down gasping for breath after he was hit on the band of his shorts.
"Anthony and Daniel, I know the IBF title is important to you. It is my present to you on September 21," Usyk said on X when he vacated the belt.
The winner of the bout between Joshua and Dubois could then get a crack at the undisputed champion after Usyk and Fury have their rematch.
The undercard will have five bouts, including two for the IBF super-featherweight title and the WBO interim light-heavyweight title.
"This is probably the most stacked card in British boxing history, certainly in my lifetime, headlined by Daniel Dubois and Anthony Joshua," Queensberry promoter Frank Warren said.
"Where better to do it at the national stadium, Wembley?"
