A panel of three Supreme Court justices in London have ruled that the case does not raise “an arguable point of law”.
The court announced its decision to refuse permission to appeal in a statement issued on Friday.
The IndyCamp group set up a collection of caravans and tents outside Holyrood towards the end of 2015 and vowed to stay there until Scotland became independent.
It kickstarted a court battle as the Scottish Parliamentary Corporate Body (SPCB) sought to remove them from the grounds.
Judges at the Court of Session in Edinburgh ruled in favour of the parliament last summer, and again later on after an appeal, paving the way for the eviction of the campaigners.
Despite being removed from the site, the group continued its legal battle.
In their latest move, campaigners applied to the UK’s Supreme Court in a bid to take their case further.
But the justices refused permission for an appeal after “a review of the relevant written submissions”.
Campaigners had argued that eviction would infringe their rights to freedom of expression.
During previous court hearings they put forward several arguments against eviction - including claims that Jesus Christ had given permission for the camp.