The prime minister has come under intense pressure from Tory MPs in recent weeks over the UK's membership of the EU, with many pushing for a referendum as sooner than his preferred 2017 date.
"It's a very clear, very decisive policy. Let me say, this policy - it doesn't matter the pressure I come under from outside the Conservative Party or in Europe or inside the Conservative Party - this policy is not going to change," he said.
He told BBC Radio 4's Today programme: "The question is not going to change, the number of referenda isn't going to change, the date by which we hold this referendum isn't going to change. The fact is it's the right policy for the country."
Cameron has pledged to hold an in/out referendum by 2017 after negotiating a looser relationship between London and Brussels - should he win the next general election.
Large numbers of Conservative MPs took the almost unprecedented step of voting to condemn the prime minister's Queen's Speech last week after it failed to include legislation that would start the ball rolling on a referendum.
And despite denials from Downing Street, Cameron was accused of being bounced into publishing a draft referendum bill in order to placate rebellious Tory backbenchers.
Writing for The Huffington Post UK, Ed Miliband said the prime minister had been "pushed around by Conservative backbenchers" on Europe so much that his policy "now appears driven by the short term political interests of internal party management rather than the long term interests of British businesses and British workers".
"The events of the past three weeks have only served to underline how distant and distracted David Cameron, not to mention his divided party, has become from addressing these issues," he said.
However Cameron insisted there was "unity" on the Tory benches behind his strategy and there had simply been "a disagreement over the last couple of weeks about an issue we actually agree about".
"If you take the issue of Europe, I think there is actually incredible unity and agreement, not just in the Conservative party but, I would argue, across the country," he said.
"That is the boldest, clearest, most straightforward policy on Europe that any party leader has had for 30 or 40 years. So it's that sort of leadership, that sort of clarity that's required."