Mr Johnson makes the commitment in the introduction to the Scottish Conservative manifesto, which carries the words "No to Indyref2" prominently on the cover.
The Prime Minister claimed Scotland had been "paralysed" and "trapped like a lion in a cage" by Nicola Sturgeon and the SNP refusing to accept the No result of 2014.
He said stopping Indyref2 would "restore confidence and certainty to businesses and families", with politics returning to priorities such as public services.
He wrote: "If the outcome of this election is a strong Conservative majority Government - then I can guarantee that we will reject any request from the SNP Government to hold an independence referendum."
Referring to Ms Sturgeon requesting a Section 30 order to give Holyrood the power to hold a legally watertight referendum, Mr Johnson went on: "There will be no negotiation - we will mark that letter return to sender and be done with it.
"You have already made your decision five years ago, when two millions Scots said no to Independence. Nicola Sturgeon promised you this was a once-in-a-generation decision and I will hold her to that."
The comments contradict those of his Scottish Secretary Alister Jack, who recently said a SNP majority win in the 2021 Holyrood would be a "democratic mandate" for Indyref2.
Ms Sturgeon has said she wants to hold another referendum in late 2020, but has so far failed to explain what she would do if the UK Government simply refused.
Jeremy Corbyn has said Labour would not permit Indyref2 in the early years of a Labour
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