Nathaniel Mendez-Laing, Alex Gilbey and Callum Paterson scored the goals as the Dons grabbed the win needed to confirm their promotion.
Victory secures the Dons' return to League One after three seasons away and also sees them leapfrog Bromley to go top. Tranmere remain 20th and are still yet to seal their League Two status.
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The hosts took the lead after 18 minutes. Mendez-Laing was first to an attempted clearance and thumped a first-time strike past Tranmere goalkeeper Marko Marosi.
Tranmere nearly equalised when Kaiyne Woolery missed from close range, before MK Dons extended their advantage three minutes after opening the scoring. Captain Gilbey collected Mendez-Laing's pass and curled a strike into the bottom corner.
Tranmere substitute Kristian Dennis had a goal disallowed for offside just after half-time and he had another strike denied by Craig MacGillivray.
But the hosts completed the scoring after 77 minutes when Paterson glanced in Liam Kelly's free-kick.
