The proposal from Bolton Wanderers chairman Phil Gartside to allow the two Glasgow clubs - collectively known as the Old Firm - failed to gain the support of the necessary minimum of 14 of the 20 Premier League club chairmen.
In a statement, the Premier League said the proposal to admit Scotland's biggest two clubs was "neither desirable or viable".
However, another proposal from Gartside to have a two-tier Premier League will be considered by the League's strategic review committee which is due to report by the end of next year.
Premier League chief executive Richard Scudamore told reporters after the meeting that the idea had been rejected clearly.
"The clubs discussed it and they basically said whilst they were prepared to look at all the other things in the round as part of the strategic development and things we are looking at, as regards to Celtic and Rangers it's a non-starter," he said.
"Therefore we have made a clear and unequivocal statement and we are going to move on from there. No means no.
"The clubs constitutionally voted basically to say we are not going to take this any further. Celtic and Rangers are not coming in," added Scudamore.
Wolverhampton Wanderers chief executive Jez Moxey added: "It's the English Premier League. It's our product and we are working on our product. The Premier League clubs have made their decision and we move on."
The decision was welcomed by rival clubs north of the border, however.
"From a selfish point of view, I want to take my team to Ibrox and Parkhead, I want to go to those arenas in front of their supporters," Hibernian manager John Hughes told Sky Sports News.
"Being a manager of an SPL (Scottish Premier League) side, I think they are the cherry on the icing on the cake for us. I feel for them, but I'm delighted that they are still part of Scottish football."
