The Prime Minister said a newly independent Scotland would have to "queue up" to become a member of the EU and added that it was a "source of regret" that Scottish people living in the rest of the UK would not be able to vote in September's independence referendum.
Serbia, Montenegro and Macedonia are all categorised as candidate countries by the European Union, meaning they are in the process of integrating their laws with the EU.
Cameron warned the "risks of separation are very great" as he was asked about the Scottish issue while on the campaign trail in Newark ahead of Thursday's by-election.
He said: "If Scotland vote for independence they are no longer members of the EU and it's become clearer and clearer since this campaign started that they would have to reapply to join the EU and as such, as an independent country, they would have to queue up as it were behind other countries - for instance those in the western Balkans that are already on the path towards membership.
"This is yet another reason why I very much hope that the Scottish people will vote no, will vote to remain in the United Kingdom. I think we're a successful family of nations all bringing something to this United Kingdom. Let's hope that we'll stay together. I think the risks of separation are very great."
Cameron said he was "confident" that the No campaign would win and said the issue of Europe was just one of the risks.
Listing other issues he said "The risk that outside the UK you wouldn't keep the pound, the risk that outside the UK you've got to reapply to the EU, the risk that outside the UK you wouldn't have such a strong economy with so many jobs and you know, in the end, it's a positive argument I'm making because I think the UK has been a great success story.
"We're there for each other. You know when there was a banking crisis and a big Scottish bank goes down you've got the whole of the UK there to support that bank and that economy. We're stronger and better off together.
"We obviously have great rivalry on the football field and rugby field and one or two other things but we're a family of nations and I hope we stay together."
All of the major UK political parties have now presented their devolution proposals following the publication of the Conservative Strathclyde Commission on the Future Governance of Scotland.
The Tories want to give Holyrood full control over income tax, the commission said, an advance on Labour's proposal to devolve three-quarters of the basic rate and closer to the Liberal Democrats' proposal to devolve almost all income tax powers.
Prime Minister David Cameron said he wants his party's proposals implemented soon after the 2015 general election, but recognised the need to build consensus.
Scottish National Party deputy leader Nicola Sturgeon urged Scots to shun the "desperate bidding war from the unionist parties" and vote Yes.