The study found senior university staff believe the Scottish Government has "centralising tendencies" with recent legislation "threatening their autonomy" and "likely to impede their effectiveness".
University principals and other officials also attacked new outcome agreements between institutions and the Scottish Funding Council, arguing they were counter-productive, limited freedom and caused "resentment".
The study by researchers at Edinburgh University also found that college representatives believed their autonomy had been "greatly curtailed".
The attack comes after the passing of the Post-16 Education (Scotland) Act last year, which gives ministers new powers to set priorities for universities in return for their public funding.
These include widening participation to students from deprived areas. and ensuring institutions comply with new rules on governance.
The report concluded: "The majority of senior managers believed that the Scottish Government had centralising tendencies and saw outcome agreements as a new form of governance which was likely to impinge on their autonomy.
"Taking a global view, they argued that the most successful institutions had the least interference from government."
However, a spokesman for the Scottish Government said: "Outcome agreements give greater exposure and transparency to the work our universities and colleges do. They allow us to chart the real progress that has been made in the three years since their introduction and demonstrate how the sectors are contributing to our national outcomes.
"The recently-published outcome agreements set out an ambitious agenda for the coming years and our investment of £1.6 billion into further and higher education."
The wide-ranging study by Edinburgh University also raises the controversial subject of fees for Scottish students, who currently have their tuition paid for them.
The report, which has been backed by the Economic and Social Research Council, found many respondents, including politicians and university officials, were not opposed to fees in principle and thought current policies were unsustainable.
Many university managers believed that the current funding "challenges" in Scotland "might make some form of student contribution necessary in the future".
A graduate tax and more progressive income tax were both mentioned as possible ways of raising additional funds, with concern the English system placed too high a burden of debt on students.
On the issue of independence, interviewees - including those with a civil service background - believed the government would have difficulty continuing to charge students from the rest of the UK fees because it ran counter to European law.
The future of research funding was also seen as causing anxiety, with concerns the existing UK funding bodies would cease to exist.
The Government spokesman said: "With independence it will continue to be in the interest of both Scotland and the UK to remain part of a single research area.