With less than two weeks of campaigning left before the general election, Jackson Carlaw said a further plebiscite on either Brexit or independence would cause a "corrosive and brutal" atmosphere - and claimed voters were instead looking for "the clarity of moving forward" by leaving the EU.
The Tory MSP also revealed in an interview today that if a second vote on Brexit did take place, he would now campaign for Leave - a reversal on his own previous stance.
"Since 2016, the EU itself has changed," he told BBC Radio Scotland. "It's now seeking to created a united army of the European Union by 2025, I can't support that.
"I believe in other ways, too, the European Union has changed."
Asked whether the SNP winning a majority of MSPs at the next Holyrood election would be grounds for an IndyRef2, Mr Carlaw said: "I am absolutely clear that we will not support a second independence referendum for another generation. The Prime Minister has also made that clear.
"I can tell you as as leader of the Scottish Conservative and Unionist party, that we will not betray the two million people who voted on the basis that it was a once in a generation vote in 2014.
"The First Minister herself said the basis on which we had the 2014 referendum was a gold standard. That gold standard was underpinned by the support of all the parties of the Scottish Parliament for a section 30 order, not a partisan alliance of two parties.
"There are variables. I was asked this question recently, and I noted the difference between the two referendums on the EU was 40 years. That seemed a fine definition to me."
Mr Carlaw was also asked about his support for Boris Johnson on the radio programme. He said he would not judge the Prime Minister on comments he has made in the past but on his record as in Number 10.
Mr Johnson has come under fire for a number of comments made in newspaper and magazine columns, written before he took office.
He previously compared women wearing the niqab to "letter boxes" and "bank robbers", as well as other remarks deemed to be derogatory towards black people and members of the LGBT community.
Asked about his views on the comments, Mr Carlaw said: "When I saw the Prime Minister before the election, I said to him what I said on broadcast media previously, which is that I'm going to judge the Prime Minister on what he says and does as Prime Minister, not what he said before he was Prime Minister."
Mr Carlaw said he would not use the terms Mr Johnson has been criticised for using. He added: "The specific things that I have asked - that he put the union at the heart of this campaign, that he respond to specific issues that we face here in Scotland and that he put them in the manifesto, that is what he has done.
"Therefore, in the sense that he has been absolutely straight in his dealings with me, then I have been able to support and continue to support him as Prime Minister."