Chris and Colin Weir donated £500,000 each to the SNP in February, according to the elections watchdog the Electoral Commission.
The Weirs previously gave the party £1 million in September 2011 and have also funded the Yes Scotland campaign to the tune of £3.5 million over the last two years.
The latest figures, which show donations received during the first three months of this year, will place further pressure on Ed Miliband following a difficult week for the Labour leader.
Two polls in recent days have shown the Conservatives establish a lead over Labour, while the party is also outstripping its opponents in fundraising.
The Tories received £6,662,232 in total, over a fifth of the money, £1,506,500, from a single donor - British-Australian hedge fund boss Sir Michael Hintze.
Labour received £4,409,167, with two-thirds of its cash coming from three of the UK's largest unions Unite, Usdaw and Unison.
But Labour later said that the true total figure was actually much higher, at £8.2m.
The extra was made up from small donations and membership fees which were not recorded by the Electoral Commission, the party said.
Donations to the Liberal Democrats totalled £1,074,482.
Scottish Conservative chief whip John Lamont said the SNP was "clearly embarrassed" that such a proportion of its funding came from one couple.
"The fact is, if the Weirs hadn't picked their lucky numbers, the independence movement would be millions of pounds worse off."
An SNP spokeswoman said: "We thank all of our donors - big and small - for contributing to the success of the party, including consistently being ahead in the polls after eight years in government, which is a remarkable achievement."
Tory chairman Grant Shapps attacked Labour's reliance on union donations.
But Labour hit back saying that half of all Tory donations had come from those who had had donor dinners either with the Prime Minister or one of his ministers.
Meanwhile, Labour's new election guru, the former Obama aide David Axelrod, discussed Scottish independence after his first meeting with the party's shadow cabinet on Thursday.
The one-time White House staffer chatted about the referendum with shadow Scottish secretary Margaret Curran.
Regarded as the architect of the American president's election victory in 2008 Mr Axelrod is understood to have emphasised how important he considers September's vote.