Nabil Karoui, 56, was detained last month on charges of money laundering and tax fraud, which he denies.
The media mogul was still able to stand despite his arrest but he was not allowed to vote. He reached the run-off vote which is expected next month.
On Wednesday, a judge refused to rule on whether or not he would be released.
The judge said the matter was not in his jurisdiction. It is the third time he has had an appeal turned down.
Mr Karoui, who began a hunger strike earlier this month to demand his freedom, will remain in jail.
His lawyer, Kamel Ben Messoud said his client would challenge the court’s decision.
Mr Karoui has never held political office. He founded a charity focused on fighting poverty and that issue has been a central theme of his campaign.
Critics have accused him of using the charity and his TV channel to further his political ambitions.
The result marked a blow for Tunisia’s political establishment, including for Prime Minister Youssef Chahed and former interim President Moncef Marzouki, who both failed to progress.
The winning candidate will be appointed to office for a five-year term.
Tunisia’s president has control over defence, foreign policy and national security. The prime minister, chosen by parliament, is responsible for other portfolios.
Separately, parliamentary elections are also scheduled to take place next month.
This election is the second free presidential poll since the 2011 uprising that toppled ex-President Zine al-Abidine Ben Ali and sparked the Arab Spring.
It was brought forward after the death in July of the country’s first democratically elected president, Beji Caid Essebsi, who took office in 2014.