St Andrews University, in Fife, founded in 1413, came top in Scotland and sixth in the UK in the 2014 Complete University Guide.
Edinburgh University came second in Scotland after falling two places to 18th in the UK table while Glasgow University dropped from 17th last year to 23rd.
Strathclyde and Dundee both fell four places coming 41st and 47th respectively.
In separate listings, Edinburgh University appeared in top 10 listings for 25 out of 48 subjects – and came top for nursing and social work. Glasgow came top for veterinary medicine, while Strathclyde and Robert Gordon, in Aberdeen, both led one subject listing.
Across the UK, Cambridge came top in the rankings, with Oxford returning to second place after being overtaking by the LSE last year.
Professor Louise Richardson, principal of St Andrews University, said: "This result is clear evidence of the dedication, engagement and academic rigour of everyone who works and studies at St Andrews."
A spokeswoman for Universities Scotland, which represents university principals, added: "All have their unique strengths at subject level and different personalities so anyone considering university should take the time to find an institution that will be the right match for them."
Dr Bernard Kingston, principal author of the rankings, said: "The lower end of the ranking should be treated with some discretion, but what is beyond dispute is that the top 10 includes some of the world's finest higher education institutions."