The monarch's words - which were interpreted by some as offering encouragement to Unionists without leaving her open to accusations of political meddling - came as the long campaign entered its final week, with tensions high and tempers increasingly frayed.
Salmond predicted that the UK Government would drop its opposition to a eurozone-style currency union with a separate Scotland in the days after a Yes vote.
This prompted Alistair Darling, leader of the pro-UK Better Together campaign, to argue that Salmond was performing a "premature victory lap" before Scots had even gone to the polls and warned his plan relied on the UK Government "falling into line" with his wishes.
Both men appeared on BBC One's Andrew Marr Show as a series of opinion polls gave contradictory views of who is winning.
Salmond confirmed the referendum would settle the independence matter for a generation, citing as an example of the minimum interval the devolution referendums of 1979 and 1997.
Challenged by Marr unequivocally to confirm there would not be a referendum sooner, he said: "That's my view. This is a once-in-a-generation opportunity, perhaps even a once-in-a-lifetime, opportunity for Scotland."
If the separatists win, the First Minister said he would start bringing together his "Team Scotland" negotiating team on Friday and he knew who he would invite to join it.
He said the list would include Darling, former Prime Minister Gordon Brown and Alistair Carmichael, the Scottish Secretary.
But Darling said: "He seemed to be doing a rather premature victory lap even before most of us have voted but he was simply asserting lo and behold on Friday morning everybody else is wrong.
"They are all going to fall into line with what he is going to say."
Darling said a No vote would mean a stronger Scottish Parliament, citing a fast-track timetable for delivering extra powers that was disclosed by Brown last week.
In Glasgow yesterday, thousands of angry Yes supporters waving saltires mounted a protest outside the BBC's Scottish headquarters, accusing it of being biased in favour of retaining the Union.
Darling said he had been shocked by the aggressive behaviour of some nationalists, which he claimed had been "orchestrated" and "centrally controlled".
Prime Minister David Cameron was hoping to avoid a hostile reception when he returned to Scotland today in a last-gasp attempt to persuade Scots to preserve the Union.
The Queen made her comments while in conversation with a churchgoer outside Crathie Kirk in Aberdeenshire, where the Royal Family worship while they are in residence at nearby Balmoral Castle.
Royal sources said she had given a "spontaneous" response to a question she was asked after the service, adding that "the words speak for themselves".
It is understood that reporters were invited to listen to the Queen's exchanges with the public, which she usually prefers to keep private. Buckingham Palace said it did not comment on "private conversations" held by the monarch.
"The Queen has made it very clear that the referendum is a matter for the people of Scotland," a spokeswoman added.
Yes Scotland said the Queen was "echoing the message" it was sending voters to "think very carefully about this one opportunity that Scotland will have on Thursday to choose our future", while a Better Together source merely said she had been "very clear that she doesn't want to comment" on a democratic decision.
- Independent
Judgment day
The question
"Should Scotland be an independent country?" to be marked with an X for 'Yes' or 'No'
Polling stations
Open at 6pm Thursday and close at 9am Friday NZT.
Results
From the Shetland Islands in the north to Edinburgh and Glasgow in the south, from the Highlands in the west to the oil hub of Aberdeen in the east, results from 32 local authorities will emerge.
Turnout
It is expected to be a record high and the official result will be announced in Edinburgh on Friday night NZT.
Voters
Nearly 4.3 million people have registered - 97 per cent of the electorate - indicating the level of interest in a vote which will include 16 and 17 year olds for the first time.
Celebrity backers
Yes
Actor Sean Connery, comedian Russell Brand, film director Ken Loach, author Irvine Welsh, designer Vivienne Westwood and actor Peter Mullan.
No
Author J.K. Rowling, football star David Beckham, singer Paul McCartney, singer Mick Jagger, astrophysicist Stephen Hawking, actress Helena Bonham-Carter, actor Michael Douglas, actress Judi Dench, comedian Mike Myers, actress Emma Thomson, singer David Bowie.
Keeping quiet
Actor James McAvoy, tennis star Andy Murray.
- AFP
