The debt-ravaged club face a winding-up petition from the Government's Revenue and Customs office in the High Court next month and had appealed for permission to sell players in a bid to raise urgent funds.
However, the Premier League has declined their request.
"The Premier League board can confirm they have been actively considering a request from Portsmouth to transfer players outside of the transfer window," the League said in a statement.
"We are grateful for positive assistance from FIFA and the FA but, having given the matter further consideration and taking into account all factors, the Premier League board have decided that this would not be an appropriate course of action at this time."
Portsmouth's application for the unprecedented move was forwarded by the Premier League to both the English FA and to FIFA, world football's governing body, but manager Avram Grant said on Friday he did not want any of his players sold.
Speaking ahead of Saturday's Premier League match against Stoke, Grant told a news conference: "I don't even want to talk about this because I came to this club to do something. I came here to save this club from relegation.
"But every week, or every day sometimes, there is a new thing and I don't know whether these things are sad or funny.
"We got a good result against Southampton and I thought nothing could happen now because there is no transfer window. You cannot say there is no creativity at this club."
West Ham United co-owner David Gold said on Friday he was prepared to lend Portsmouth 10 million pounds, underwritten by the Premier League, to save them from being liquidated.
South coast club Portsmouth, who are bottom of the Premier League table, are due back in the High Court on March 1.
