Opposition parties warned that confident Nationalists are “on manoeuvres” for a second vote on leaving the UK.
The SNP leader came under pressure to rule out another vote as she faced MSPs for the final time before the election at First Minister Questions yesterday.
Ms Sturgeon had claimed during the referendum campaign last year that it was a “once in a generation” event but has since claimed that a change in “material circumstances” could prompt a return to the polls.
But the Nationalists are poised to take up to 50 of Scotland’s 59 seats in the election and many SNP candidates have indicated they want to see another quickfire vote on the constitution.
Liberal Democrat leader Willie Rennie said Scots have had enough and demanded an end to the constitutional wrangling from Ms Sturgeon.
“She knows that a vote for her party is a vote for the second referendum party. Can she now, finally, at last, rule out a second referendum for a generation?”
Ms Sturgeon declared in an interview this week: “I am not going to rule it out.”
But Mr Rennie added: “That’s not what she said before last September. Now she expects people to believe her this time.
“We know her colleagues are on manoeuvres for a second referendum but the First Minister can sort this out now.
“She has to show leadership on this. Will she rule out serving as First Minister in a Scottish Government that holds a second referendum?”
Ms Sturgeon has insisted that this Westminster election won’t provide a mandate for another referendum.
“I am very grateful to Willie Rennie for giving me the opportunity again to directly address the people of Scotland on this issue,” she said.
“This election tomorrow is not about independence. Even if, and I am not making a prediction here, the SNP wins every seat in Scotland that is not a mandate for independence or a second referendum.”
She added: “Willie Rennie is clutching at straws here. I am happy, as we are right now less than 24 hours before the opening of polls in this unique, perhaps watershed election, to let the Scottish people have their say.
“I am very clear what this election is about and is not about, and I will let the people of Scotland judge. This election is not about independence.”
The issue is likely to return at the SNP’s autumn conference when a decision will be made on whether to include a commitment to a second referendum in the party’s manifesto for the Holyrood election next year.
Former party deputy leader Jim Sillars said recently it should be in the first line of the manifesto if the party make sweeping gains at the Holyrood election. If the SNP won another Holyrood majority with a pledge to stage a second referendum, Scots would face another vote on leaving the UK in the next few years.
Scottish Tory leader Ruth Davidson had earlier appealed to No supporters as she warned that every vote for the SNP is a “vote that takes a step back towards another referendum” in a keynote speech yesterday.
She said: “We made a really big decision in September of last year about what form we wanted our country to take and we voted to say we wanted to be part of the UK that had a UK government.”
Scottish Labour’s deputy leader Kezia Dugdale also warned that big SNP gains today will result in a second referendum.
“Nicola Sturgeon won’t rule out a second referendum because she knows a vote for the SNP in the general election will send Scotland on the road to one,” she said.
“We’re at a crossroads in the future of our country. A vote for the SNP sends us back to the same old arguments about the constitution, a vote for Labour sets us on the road to a fairer Scotland with a Labour government.”
Ms Dugdale insisted there would be “more progressive policies in the first week of an Ed Miliband government than in eight years of an SNP government’’.
Ms Dugdale said the SNP have sided with the Tories in the Scottish Parliament to defeat Labour plans on the living wage, banning zero-hours contracts and ending “rip off” rent rises.