Leaving the UK would create "a number of costs and uncertainties" for business with "fewer, more uncertain benefits", according to a report commissioned by the Scottish engineering firm Weir Group.
The study, commissioned by one of Scotland's largest companies, says the country's economy "could succeed" if there is a Yes vote in September's independence referendum.
But it goes on to warn: "The end of the Union would create a number of costs and uncertainties, and fewer, more uncertain benefits, for those businesses so vital to Scotland's future prosperity, as it goes its own way."
Weir Group, which employs 600 staff in Scotland and 15,000 people globally, commissioned Oxford Economics to carry out the report
The impact on businesses of Scottish independence "could be substantially negative" with monetary conditions, funding costs, and transaction costs "only likely to change for the worse under independence," the authors suggest.
It has been published in response to the Scottish government's white paper on independence, and seeks to address the issue of what leaving the UK would mean for firms north of the border.
The report argues a "key benefit" of independence would be the "policy flexibilities that it would bring".
It says: "Independence would clearly bring control over policy making closer to the people of Scotland. It would allow a Scottish government to tailor an expanded range of economic policy levers to the needs and circumstances of the Scottish economy, as well as the distinctive views and values of the Scottish people."
But it adds there could be "risks and costs to businesses in adapting to the changes implied by independence, as well as costs that flow from the inevitable uncertainty during the transition phase that would follow a Yes vote".
The report describes the Scottish government's estimate of the 27,000 jobs that could be created by cutting corporation tax as being "probably over-optimistic".
It goes on to warn that "fiscal realities will likely constrain the policy choices of an independent Scotland", arguing that "an independent Scotland is likely to need to tax Scottish business overall more heavily than if it remained in the UK".
Whatever currency arrangements are established, it fears Scottish companies are "likely to face higher funding costs post-independence".
Meanwhile if an independent Scotland cannot reach a deal to keep the pound in a currency union - something the main UK parties have already rejected - it says businesses will face additional costs if the country is forced to adopt its own currency after independence.
Changing currency could mean "substantial one-off costs for business, amounting to around £800 million", according to the report, along with "ongoing transaction costs on Scottish businesses and households of around £500 million per year".
In addition it says independence would add "new complexities to businesses in providing pensions for their employees".
Weir Group is the latest to add its voice in warning against Scottish independence, including the following business leaders:
