Douglas Beattie, who is standing in Dumfriesshire, Clydesdale and Tweeddale, urged Labour members to vote Yes in 2014.
The Scottish Conservatives highlighted his comments after Labour's Alistair Darling attempted to shore up his party's shaky position on Nicola Sturgeon's demand for a second referendum.
David Mundell, who is defending the DCT seat for the Scottish Conservatives, said Labour were in chaos on the issue.
Mr Beattie - a councillor in the London borough of Camden - posted a series of tweets backing the Yes side in the 2014 campaign.
After the vote, he said he was "gutted for Yes camp who fought so justly and valiantly".
He also declared there was "something of the undertaker" about Mr Darling and criticised his leadership of the Better Together campaign as "fearty max".
Labour have sent mixed messages about their support for the Union.
Leader Jeremy Corbyn has said he is "absolutely fine" with the SNP holding a second referendum.
Before the EU referendum, Scottish Labour leader Kezia Dugdale said it was "not inconceivable" that she might support independence in the event of a Leave vote.
Scottish Conservative candidate for Dumfriesshire, Clydesdale and Tweeddale, David Mundell, said:
“Yet again Labour are in chaos over the threat of a second independence referendum
"They are trying to present themselves as solidly pro-Union but one of their own candidates backed independence.
"But no-one should be fooled after Jeremy Corbyn said he is ‘absolutely fine’ with the SNP holding a second referendum.
"Only the Scottish Conservatives will stand up to Nicola Sturgeon's obsessive push for a second unwanted, divisive vote on taking Scotland out of the UK.”