In July last year, BT and TalkTalk called for the High Court to launch the review, stating the Act was "rushed through" and had "insufficient scrutiny". The two firms were also concerned that measures to tackle net piracy - including plans to temporarily suspend people from the web - could be in breach of "basic rights and freedoms".
However, the judicial review, which began in March this year, rejected four out of the five claims put forward by the ISPs. Last month, the pair said they wanted those four grounds looked at again.
"Both companies believe the High Court's conclusions on many of the other important and complex issues put before it were not robust enough to provide the certainty and clarity which the companies sought," the ISPs said.
However, Judge Sir Richard Buxton refused the application this week. The ISPs have also been ordered to pay around £100,000 of the legal fees incurred by the judicial review.
A spokesman for the two ISPs told The Guardian: "We are now considering our position."
Tony Ballard, a digital media specialist at media and entertainment law firm Harbottle & Lewis, said: "If it [the refusal of an appeal] really marks the end of the litigation, its significance lies in Ofcom being now able to implement the procedure for discouraging peer-to-peer file sharing under the Digital Economy Act without the cloud of uncertainty as to the validity of the Act hanging over it. Opinion in the industry remains divided, however, as to whether the procedure is a good idea or not."