Promoter Eddie Hearn says Jarrell Miller has failed a second doping test and as such a new opponent for Anthony Joshua will be announced next week.
Miller said on Thursday he had "never knowingly taken any banned substance" and intended to appeal against the decision to revoke his licence following an adverse doping test.
Miller and Joshua were set to meet in New York on June 1 and Hearn says he has now learned of a second alleged doping violation by Miller, meaning that fight is now dead in the water.
"We have now received news from VADA that Jarrell Miller has now failed a second separate test for a further substance. AJ's June 1 opponent will be announced next week – if you don't think you can beat him clean then delete my number!" he wrote on Twitter.
VADA has been contacted for a response to Hearn's claim.
Speaking on Thursday, Miller said: "I am absolutely devastated upon hearing the news my boxing licence has been revoked in NY State and I will be vigorously appealing this decision.
"I have NEVER knowingly taken any banned substance and when I found out the news last night I was totally shocked.
"My team and I stand for integrity, decency and honesty and together we will stand to fight this with everything we have!"