PA MEDIA
Marcus Ball accused the prime minister of "abusing public trust" by claiming the NHS could get £350m extra a week after Brexit.
The High Court threw out his case in June - but Mr Ball applied for leave to appeal to the UK's highest court.
On Wednesday, Lady Justice Rafferty rejected his application.
The proceedings were started at Westminster Magistrates' Court in May when Mr Ball applied for a summons accusing Mr Johnson of three allegations of misconduct in public office.
Mr Johnson's lawyers have always rejected claims he acted improperly or dishonestly in any way and challenged the case at the High Court.
Mr Ball could still continue his legal battle by applying directly to the Supreme Court, challenging the decision in the European Courts or making another attempt at a prosecution.
"This isn't over, we are not giving up," he said outside the High Court after the ruling. "We are pursuing it, absolutely."
He later said in a statement: "The offence of misconduct in public office has itself never come before the Supreme Court and is in great need of their attention and clarification."
The £350m figure was used by the pro-Brexit Vote Leave group during the referendum.
It also appeared on the side of a campaign bus, which urged the UK to "fund our NHS instead".
The claim was criticised by, among others, the UK's statistics watchdog.
Mr Ball crowdfunded more than £390,000 for legal fees to fight the case, arguing: "We do not send that amount of money to the EU each week.
"We have evidence that Mr Johnson knew that we don't."
He sought to prosecute the Tory MP and ex-Mayor of London on three counts of misconduct in public office.
However, the High Court said last month it was not proved Mr Johnson had been acting in a public office.
Lady Justice Rafferty - one of the the High Court judges who threw the case out in June - made a brief announcement on the decision to block the case from the Supreme Court on Wednesday.
"This application for leave to appeal to the Supreme Court is rejected," she said.
https://www.bbc.com/news/uk-politics-49345223
