David Mundell said the majority Scots don't want and another vote on leaving the UK and pledged to stand up against the SNP's proposals at the Tories' Scottish Spring conference in Aberdeen.
The First Minister announced last week that she will bring forward plans at Holyrood for a second vote on leaving the UK in response to the Brexit turmoil and indicated the vote will happen next year.
But Westminster has control over the constitution and Mr Mundell warned today there will be no section 30 which was agreed in 2012 to pave the way for the last referendum.
"We in the Scottish Conservatives know that we represent a majority of Scots when we tell the SNP that they should abandon their plans for a second independence referendum," Mr Mundell said.
“But let me be clear: if Nicola Sturgeon continues with her Indyref2 plans and asks for the UK Government’s agreement to hold a referendum, the answer will be No!
"No Section 30 Order. No second Edinburgh Agreement. No legal referendum.
"The people of Scotland said No in 2014 and they meant it!
“Ruth and the Scottish Conservatives want nothing more than to debate the future of Scottish education and how to grow our economy.
"Yet while the SNP are consumed with their constitutional obsession we must continue to stand up to them."
The SNP changed policy last weekend to pave the way for a separate Scottish currency after a Yes vote, but this was branded "chocolate money" by Mr Mundell.
"We know the damage independence would do. For example, it would take £10 billion out of our public services - real money, not chocolate money!"