Keith Brown made the claim in the same week the Supreme Court ruled the Scottish Parliament cannot legislate for another independence vote without express consent from Westminster.
Nicola Sturgeon has now reiterated her plans to treat the next general election as a de facto referendum.
Speaking to the BBC on Sunday, Mr Brown said the UK Government fears it could lose Scotland if it accedes to the demands of the independence movement.
He said: “I think they know they’re going to lose this, that’s why they are doing everything they can to twist democracy, to refuse the opportunity for the people of Scotland, because they know they’re going to lose.”
Mr Brown pointed to a snap poll by Find Out Now for Channel 4 of 1,006 Scots, which suggested 51 per cent would vote for the SNP if they knew their vote would be used to negotiate independence.
He added: “(The UK Government are) scared, that’s the point.”
Mr Brown also sought to tamp down talk of dissolving Holyrood and using the subsequent election as a de facto referendum, which had been posited by SNP MP Angus MacNeil following the Supreme Court ruling.
To force a Holyrood election, two-thirds of MSPs would have to vote in favour or the post of first minister would have to be vacant for 28 days.
Mr Brown said: “We do want to have a referendum next year, and we could do that still if the UK Government just agreed to the proper route they’ve agreed in the past.
“That’s the reasonable way to do it, that’s the democratic way to do it.”
Ms Sturgeon has previously said she is willing to speak to Prime Minister Rishi Sunak about the possibility of a Section 30 order which would grant Holyrood the necessary powers to stage a vote.
The Scottish Government has said it accepts the outcome of the Supreme Court case, but stressed “Scottish ministers remain ready to engage with the UK Government at any point on delivering that referendum.”
Speaking immediately after the verdict, a spokesman added: “The Scottish Government will continue to set out, through the Building a New Scotland prospectus series, what could be done with the full powers of independence, reflecting clear Programme for Government commitments.”
The depute leader’s comments come after he said the Yes movement would hit “new heights” following the Supreme Court decision, which he claimed “shattered forever the notion of the UK as a voluntary union of nations”.
He added: “It also laid bare the duplicity of the Westminster parties who are flagrantly breaching their own pledges to the people to respect Scottish democracy.
“But if those same parties think that this week has ended the debate on Scotland’s future, they couldn’t be more mistaken.
“It is a movement which will hit new heights by galvanising public opinion in every city, town, village and community the length and breadth of the country.”
Responding to his claims, Scotland in Union dismissed the idea of a “so-called ‘de facto referendum’.
She said: “Keith Brown doesn’t get to decide the issues on which people vote for in a General Election.
“Rather than trying to dictate the terms of a democratic election, the SNP and the Greens should focus on what really matters – the cost-of-living crisis, the emergency in our NHS, and the climate disaster.
“It’s time for the people’s priorities, not the SNP’s”.
It came as new figures showed more than £1.5 million of public money will be spent annually on civil servants who have been tasked to work on the independence campaign.
Data obtained by the Scottish Conservatives using freedom of information legislation shows there are 25 officials working on the prospectus.
Using the maximum annual salary of civil servants in each of the pay bands, the Tories calculated the Scottish Government will spend a total of £1,532,664 on staff working on the independence prospectus.
One official, a senior civil servant, is in the highest pay band where the maximum salary is £83,233, the figures show.
Five officials earn less than £47,000 on the lowest two pay ranges, followed by one who is paid £47,485 each year.
Following that, nine staff fall within the C1 pay band up to £62,167, eight civil servants are on £75,341 and one earns £77,340.
Scottish Conservative constitution spokesman Donald Cameron condemned the Government for using taxpayers’ money on its “pet project”.
He said: “Most Scots will be appalled that Nicola Sturgeon is squandering huge sums of public money and civil service resources on her pet project at the same time as imposing savage cuts on key public services.
“It is further proof, if any were needed, that the SNP leader always puts her party’s interests before those of the country.”