Those in favor remaining a part of the United Kingdom led the secessionists approximately 55 percent to 45 percent, according to official results on Friday, with 31 of 32 constituencies reporting.
The average turnout was a record high of more than 84 percent.
By 5:00 a.m. local time (0400 UTC), only four areas had voted in favor of independence.
The "Yes" campaign had pegged its hopes on a decisive victory in the largest constituency of Glasgow, but only managed to win 54.4 percent to 46.5 percent.
In the council area of Edinburgh, meanwhile, 61.1 percent of voters rejected independence.
Highland is the only constituency yet to declare, but the results cannot change the outcome of the referendum.
British Prime Minister David Cameron said that Friday's result had settled the debate over Scottish independence "for a generation."
"Now is the time for our United Kingdom to come together and to move forward," he said during a speech at 10 Downing Street.
Triumph for the 'democratic process'
The pro-independence Scottish National Party leader and First Minister Alex Salmond conceded defeat shortly after the final results were announced.
"I accept that verdict of the people and I call on all of Scotland to follow suit in accepting the democratic verdict of the people of Scotland," he told a crowd of his supporters in Edinburgh.
"The process by which we have made our decision as a nation reflects enormous credit upon Scotland," Salmond added, noting the election's high turnout. "This has been a triumph for the democratic process and for participation in politics."
Alistair Darling, who led Scotland's Better Together campaign against independence, congratulated his supporters following what he called a difficult campaign "that has both energized and divided."
"We've made a decision for the United Kingdom and for Scotland. C'mon Scotland, let's get on with it together," he said.
dr/jm (AP, AFP, Reuters, dpa)