The Prime Minister has said he sees "no reason" to ever permit a second independence referendum to take place.
Boris Johnson indicated he would not agree to a Section 30 order for a fresh independence vote even if the SNP returns a majority of Scottish MPs on December 12 or wins a majority of MSPs at the next Holyrood election.
Johnson claimed Nicola Sturgeon's party has done a "shady deal" with Labour to prop up a Jeremy Corbyn-led government in exchange for indyref2 in 2020.
Labour has ruled out agreeing to a second referendum in the "early years" of a Corbyn government although says it ultimately would not stand in the way of one if the Scottish people wanted it.
The party has also said it has done no deal with the SNP, and is not seeking pacts or coalitions with any other parties after next month's election.
The PM arrived in Scotland for a campaign stop at a whisky distillery in Moray, where he said he sees the benefits of the union "everywhere I go".
But the Scottish Liberal Democrats accused him of "undermining the integrity" of the UK with his Brexit deal, while Sturgeon demanded Johnson apologise for the "chaos of Brexit".
Scottish Labour said Johnson's trip north of the border "smacks of desperation".
The Conservatives won 13 Scottish seats from the SNP in the 2017 snap election, with the nationalists finishing runner-up in each of them.
Speaking to STV News on Thursday, the Prime Minister said the first independence referendum in 2014 had been a "once-in-a-generation event", adding: "I see no reason to back on that pledge that we made to the people of Scotland."
Pressed by STV's political correspondent Ewan Petrie on if he could continue to sustain that position irrespective of the SNP's electoral performance, Johnson replied: "I tell you what.
"I believe passionately in the United Kingdom, I think that the union between England and Scotland is one of the great political achievements of the last 300 years...
"Jeremy Corbyn has done a deal with the Scottish Nationalist Party who would sustain him in power, who would get him into power, in exchange for agreeing to another referendum even though the last one was meant to be a once-in-a-generation event.
"It's only Scottish Conservatives who are actually saying the union is a fantastic thing.
"Everywhere I go I see the benefits of the union.
"Look at the Glasgow COP26 summit that's going to happen next year, it's going to be a fantastic thing for the whole of the UK, for the whole of Scotland.
"It is coming to Scotland because Scotland is part of our wonderful United Kingdom and that's the way I think it should say."
Asked again if it would be democratic to continue to deny voters in Scotland a second referendum regardless, the Prime Minister said: "We had a big exercise of democracy in 2014."
The Liberal Democrats claim they are the "only party standing up for Scotland's place in the UK".
Scottish Lib Dem leader Willie Rennie said: "Boris Johnson is undermining the integrity of the United Kingdom by backing a deal that puts a border down the centre of the Irish sea.
"He rejected that last year but now he thinks it's good enough.
"Voters in Scotland cannot trust the Conservatives. They have put Brexit before the union.
"The Liberal Democrats are the only party standing up for Scotland's place in the UK and the UK's place in the EU."
Addressing Johnson's visit during First Minister's Question on Thursday, the SNP leader said she was "confident" of the Prime Minister's unpopularity in Scotland.
Sturgeon said: "He should take the opportunity to apologise for Tory austerity, for Tory welfare cuts, for the mess and chaos of Brexit and for the misery that has been heaped on Scottish people by the Tories for too many years now.
"I'm so confident that Boris Johnson will not go down very well with Scottish voters that my words to him as he leaves Scotland this afternoon may be: haste ye back."
Scottish Labour leader Richard Leonard said: "This trip to Scotland today by Boris Johnson is a futile attempt to save Conservative seats in Scotland and quite frankly smacks of desperation."
"Johnson represents a dangerous strain of nationalism and that is what poses the biggest threat to the future of the UK. They have nothing to offer but more cuts and more division."
"The people of Scotland have it in their power to banish Johnson and the Tories from power and elect a Labour government that will invest in Scotland's public services and transform our economy."