The former Motherwell Football Club chairman warned a Yes vote in next year's referendum would create a barrier to business.
He said: "I am unashamedly against an independent Scotland. I am Scottish, live here and love the country but I think it would be an economic and social catastrophe of epic proportions to disengage with England and the Union."
He added: "If you put a barrier to people you are doing business with, that creates problems. It looks to me the chances of the majority people voting for [independence] are getting slimmer."
His remarks, in an interview with The Herald, follow comments at the weekend when he dismissed the Scottish Government's bid for independence as a "bizarre experiment". He stressed, however, he respected individuals within the SNP.
Mr Boyle is one of Scotland's most influential business figures. He built up Direct Holidays before selling to Airtours for £80 million and creating private equity vehicle Hamilton Portfolio, which has a number of business and property interests.
His comments come after Jim McColl, Clyde Blowers chairman and chief executive, backed independence, likening it to a "management buyout".
A spokesman for the pro-UK Better Together campaign said: "Scottish firms sell more to England, Wales and Northern Ireland than they do to all of the other countries of the world combined.
"We are a single market and our companies don't need to worry about boundaries or borders. There is nothing to be gained by throwing up barriers within the United Kingdom. John Boyle is the latest business figure to make this point."
A spokesman for the pro-independence Yes Scotland campaign said:"As Jim McColl, one of Scotland's most successful entrepreneurs, has said, a different approach is needed if we are to make Scotland the kind of successful, prosperous and fair country we all know it can and should be – and that approach is independence."