The Prime Minister was asked on Wednesday if he should allow a referendum if people in Scotland voted for pro-independence parties.
Refusing to directly say no, Mr Johnson suggested it was best to “wait and see what actually happens”.
He said: “I think that most people in Scotland, most people around the whole of the UK, feel that this is not the time, as we’re coming forward out of a pandemic together, this is not the time to have a reckless, and I think irresponsible, second referendum.
“We had one only a few years ago. I think what most people want is to focus on the country and taking it forward and rebuilding our economy and getting people into work.
“That seems to me to be the priority.”
For Scotland to hold a legal referendum, Westminster would have to pass a section 30 order allowing a vote to go ahead.
The Prime Minister has repeatedly ruled out doing so, despite the Scottish Tories tweeting this week that an SNP majority was a “guarantee” of a second referendum.
Asked what success would look like for the Conservative Party in the local elections across the UK, the Prime Minister insisted it would be “very tough”.
He explained: “I think when we stood last time for many of these council seats we were at a particularly high watermark and we’ll be fighting for absolutely every vote.
“The choice I think is clear – between Labour opposition who seem absolutely determined to play political games and government which is getting on with our agenda, getting on with people’s priorities – for instance, with the roll-out of the vaccine."
Kirsten Oswald MP, the SNP deputy leader at Westminster, claimed Mr Johnson's position was not sustainable.
She said: "Actually it is not for the Prime Minister or for anyone else to decide the future of Scotland should be.
"I think that whilst the Prime Minister may like to talk tough, the reality is that he won't. You won't be able to stand in the way of the people in Scotland making that choice for themselves."
It came as the former Prime Minister Gordon Brown claimed iff the SNP could not solve problems in Scotland over the past 14 years, they should stand aside for the Labour Party.
Speaking at a drive-in rally of supporters in Glasgow, Mr Brown talked up new Labour leader Anas Sarwar while urging voters to turn out for the party on Thursday.
He said: “If the SNP could not solve the health problem, the waiting list problem, the mental health problem, the social care problem in any one of the 14 years in government, they will never solve the problem now.
“That’s why they should give way to the Labour Party, who can do it.
“It’s not just a health crisis, it’s the education crisis.
“Every indicator that should be going up is going down and every indicator that should be going down is going up.
“If they can’t solve the education problem in 14 years in government, make way for the Labour Party who can.”