Treasury Secretary Danny Alexander has stepped up the case for Scotland remaining in the United Kingdom.
The SNP wants to hold a referendum towards the end of the five-year parliamentary term.
In a speech to CBI Scotland members he said the current constitutional uncertainty was in danger of distracting from the economic recovery.
He told the Glasgow gathering: "We are stronger together."
The SNP government has pledged to hold an independence referendum in the second half of the current parliamentary term.
Chair of CBI Scotland, Linda Urquhart, also addressed the annual dinner, saying that members were concerned about the commitment to a referendum and its timing.
She also called for the legality of any such vote to be "put beyond doubt".
But SNP Treasury spokesman Stewart Hosie accused CBI Scotland of "blundering into the constitutional debate".
The constitution is a reserved matter for Westminster, but the UK government has indicated it would not stand in the way of a vote in Scotland.
It is not clear yet what question or options might be included in any ballot.
Ms Urquhart said anything that added uncertainty was unwelcome at a time when economic pressure on business continued "to be relentless".
Lib Dem minister Mr Alexander said: "Scotland's road to recovery is intimately tied to recovery of the rest of the UK, and we must work together - politicians and businesses - to capitalise on every opportunity to support recovery.
"We are stronger together - and we are stronger when Scotland's two governments work together, pulling in the same direction for the people of this country."
He added: "We cannot allow the current constitutional uncertainty to distract or undermine this economic goal - Scotland is too important to the UK and the UK too important to Scotland for that.
"I hope that your voices - the voices of Scotland's economic future - will be heard as that debate develops."
Ms Urquhart said the SNP government's commitment to holding a referendum was "a matter for them", but added there was concern among CBI Scotland members "about the possible damage that could be done to Scotland by the uncertainties arising from this commitment and its timing".
She continued: "This is not helpful - but with that timescale committed to, I believe that business will consider it all the more important for any referendum to deliver a clear result: independence - yes or no - and no second questions which might produce an inconclusive result.
"The legality of any referendum must also be put beyond doubt - the constitution is a reserved matter - so the Scottish and UK governments must work together to ensure legal certainty and a decisive result."
SNP treasury spokesman Stewart Hosie questioned whether Ms Urquhart's remarks were representative of the business community in Scotland.
'Right to vote'
Mr Hosie, who is MP for Dundee East, said the CBI's credibility had previously been "undermined by blunders into the constitutional debate", claiming it had misrepresented members on issues related to powers for the Scottish Parliament.
He continued: "This is not the first time CBI Scotland has blundered into the constitutional debate and this latest intervention will fuel concerns that the CBI's leadership has a political rather than a business agenda.
"Given the overwhelming mandate the SNP secured at the recent elections, with broad support from businesses big and small, there must be a question over whether CBI is even speaking for its membership, let alone the wider business community."
Mr Hosie added: "We believe that the people of Scotland should have the right to vote in a single referendum agreed by the Scottish Parliament, on a published proposal, which is then implemented - exactly as was done for the two-question devolution referendum in 1997."