Boris Johnson has ruled out granting permission for a second vote on Scottish independence while he is prime minister.
Mr Johnson said his government would not give the go-ahead for another legally-binding referendum.
The Tory leader claimed the issue had been settled in a "once-in-a-generation" vote in 2014.
Nicola Sturgeon described the position of blocking another independence referendum as "unsustainable".
The Scottish first minister has already said she will request a "section 30 order" - which grants permission for a new referendum from the UK government - within days of the 12 December general election.
'Wonderful for harmony'
The SNP leader previously said she believed Labour leader Jeremy Corbyn would grant her the section 30 order if he was voted into Downing Street.
But Mr Johnson was clear he did not want to see Scots given a second vote on the country's place in the UK.
Speaking on Sky News' Sophy Ridge on Sunday, the PM said: "I think having another referendum, I don't think people in this country think referendums are very wonderful for harmony.
"We had one in 2014, the British people, the people of Scotland, were told in 2014 that that was a once-in-a-generation event."
Asked if he would grant permission for an independence referendum as prime minister, Mr Johnson stated: "No, I don't, I don't want to have one. But I don't see any reason to go back on that, on that assurance."
The Scottish government wants to put the question of independence to a new public vote in the second half of 2020 and has started the legislative process in Holyrood to achieve this.
However, for any referendum to be legally binding it needs the green light from the UK government, which has said it will not approve any section 30 agreement.
The Electoral Commission has also said it would want to assess the wording of the question for a new Scottish independence vote even if it was the same one used in 2014.
Reacting to Mr Johnson's comments, Ms Sturgeon, also speaking on Sky News' Sophy Ridge on Sunday, said: "It is not a sustainable position and it is certainly not a democratic position.
"Everyone in Scotland knows there is going to be another independence referendum and if the SNP wins this election then that sends a clear message that we want to take our future into our own hands rather than have Boris Johnson continue to impose a future upon us.
"If the SNP win this election, for any Westminster politician to seek to stand in the way of an independence referendum would be seeking to ignore the democratic wishes of the Scottish people and I don't think it will be a position that any Westminster politician will be able to stick to."
'We wouldn't stand in their way'
On the same programme Shadow cabinet member Rebecca Long-Bailey made clear Labour would not block a second referendum if the Scottish government was to push for one after the 2021 Holyrood election.
While she was clear Labour wants "Scotland to be part of Great Britain" she added: "Ultimately, what we have said is that after the next Scottish government elections, if the Scottish government determine they want to pursue another referendum and they go through the legislative process within their own government to push that forward, than as a government we wouldn't stand in their way."
The party has said it will oppose independence if there was another referendum.
Scottish Lib Dem leader Willie Rennie said Labour's comments on a second independence referendum showed Jeremy Corbyn's party could not be trusted to preserve the Union.
He said: "It's quite clear Jeremy Corbyn will break up the UK if it means he can get his hands on the keys to Downing Street."