Nicola Sturgeon has been urged to focus on the "day job" and rule out a second vote on independence as political leaders mark the anniversary of the 2014 referendum
Two years after the historic September 18 ballot, the Conservatives warned continuing uncertainty over the constitution would act as a "ball and chain on Scotland's economic prospects".
However, the SNP said the behaviour of opposition parties will only increase support for independence.
Scots voted by 55% to 45% against independence in 2014, with the most recent polls showing an increase in support but with a majority in favour of staying part of the UK.
In the wake of the vote to leave the European Union, the First Minister has included plans to consult on a draft Referendum Bill in her programme for government for the coming year.
The SNP has also launched a "new national conversation" on Scotland's future, with activists being tasked with speaking to two million people before the end of November.
Scottish Labour leader Kezia Dugdale believes the Scottish Government should focus on education, the NHS, the economy and cutting child poverty.
SNP business convener Derek Mackay said: "Two years on from the independence referendum it seems the Tories are more determined than ever to push Scotland towards an independent future.
"For three months, Tory ministers at Westminster have been meeting behind closed doors to decide Scotland's future - with the people firmly shut outside and no answers being given to the most basic questions.
"On whether the UK will be in the single market: no answer. On a new holiday tax to go to Europe: no answer. On protection of workers' rights: no answer.
"And on the biggest question of them all: why should Scotland be dragged to the EU exit door when every single area of Scotland voted to remain? No answer.
"Of course, these were the very same Tories who told the people of Scotland that only by voting No to independence could Scotland's EU membership be guaranteed.
"So, it is perhaps unsurprising that every poll conducted since June 23 has shown support for independence ahead of where it was in September 2014 - and that is before the impact of Brexit starts to hit home."
Scottish Conservative economic and finance spokesmen Dean Lockhart and Murdo Fraser have written to the First Minister over what they described as continuing uncertainty.
They wrote: "You as First Minister could provide no greater stimulus to the Scottish economy than to clarify that no referendum will take place.
"We can all have views about the impact of the UK's decision to leave the European Union.
"But the threat of far greater constitutional division over the future of our Union is needlessly imposing division and instability on Scotland at a time when certainty should be at a premium."
Scottish Labour leader Ms Dugdale said: "The in-tray for the SNP government in Edinburgh is overflowing - the attainment gap between the richest and the rest in our classrooms, an NHS in distress, unacceptable levels of child poverty, 150,000 trapped on housing waiting lists and a £15 billion difference between what Scotland raises in tax and what we spend on public services.
"With so many challenges facing Scotland's future, it makes no sense to return to the arguments of our past.
"Instead there should be a renewed focus on the day job from the SNP government."
Scottish Liberal Democrat leader Willie Rennie blasted former prime minister David Cameron over his handling of the independence referendum aftermath.
Speaking to activists at the party's federal conference in Brighton, he said: "David Cameron says he loved Scotland but everything he did after the referendum in 2014 undermined the union.
"He approved election posters that showed a Scot picking the pocket of the English. And then gambled on our future in Europe to try and keep his party together."
The Scottish Greens used the second anniversary of the referendum to launch a new campaign called No2Yes to encourage No voters who have become independence supporters since 2014 to share their stories.
A spokesman said: "In 2014 Greens played a key role in the independence referendum.
"We recognised that for many Scots it was a judgment call, and in light of the 2015 election, the prospect of decades more Tory government or the results of this year's Brexit vote, many have reconsidered their choice and many more are thinking of doing the same.
"This campaign is about giving these voters a platform to tell their stories.
"We continue to believe that independence offers the chance to create a more democratic, equal and green Scotland. By giving people the opportunity to share their journey from No to Yes, the Green Yes campaign is once again playing a critical role within the Yes movement."