The document claimed the uncertainty that would face Scotland in the event of a Yes vote, on everything from currency to membership of Europe, would be 'bad for business.'
It is backed by figures ranging from the openly anti-independence chairman of HSBC Douglas Flint to less well known figures such as such as John Michie, the boss of a north-east group of pharmacies..
It comes ahead of a visit to Scotland by Prime Minister David Cameron later this week.
It stated: "Uncertainty surrounds a number of vital issues, including currency, regulation, tax, pensions, EU membership and support for our exports around the world - and uncertainty is bad for business.
"As job creators we have looked carefully at the arguments by both sides of the debate. Our conclusion is that the business case for independence has not been made.
"We should be proud that Scotland is a great place to build businesses and create jobs - success that has been achieved as an integral part of the United Kingdom. The United Kingdom gives business the strong platform we must have to invest in jobs and industry. By all continuing to work together, we can keep Scotland flourishing."
Other backers of the letter are Andrew Mackenzie, the chief executive of BHP Billiton mining group and Sir John Grant, executive vice-president of policy BG Group, an energy firm.
Signatories include Audrey Baxter, executive chairman of Baxters Foods; Simon Thomson, the chief executive of Cairn Energy; Ian Curle, the chief executive of Edrington, the owners of The Macallan and The Famous Grouse brands, Hamish Grossart, the chairman of Artemis Investment Management.
Lord Haughey, chairman of City Refrigeration, former SSE chief executive Ian Marchant, currently chairman of oil services company Wood Group, Angus Cockburn, the interim chief executive of Glasgow-based power generation firm Aggreko and Douglas Ferrans, former chief executive of Scottish Amicable are also named along with Alastair Salvesen from the Salvesen haulage family who runs Dawnfresh Foods.
The letter is also supported by figures from the world of academia, such as Dr Brian Lang, the former principal of St Andrews University and Timothy Wright, the chief executive of Edinburgh University Press
The letter was organised by Keith Cochrane, the chief executive of the engineering firm, Weir Group, Amanda Harvie, managing director of The Harvie Consultancy, who is a former Conservative candidate and member of the party's Scottish Executive and Gavin Hewitt, the former chairman of the Scotch Whisky Association.
It was released the day after First Minister Alex Salmond was widely thought to have won the BBC TV leadership debate with Better Together leader Alistair Darling.