"Total, which is the North Sea's largest operator, has announced that they are withdrawing their application for the 8th licensing round. This creates considerable uncertainty about the 8th tender round," the ministry said in a statement.
Denmark has considered discontinuing the tender process because of the country's ambitious target of reducing greenhouse gas emissions by 70% by 2030.
An independent government adviser on climate change said in June the government should end all future hydrocarbons exploration in the North Sea because it would hurt Denmark's image as a climate front-runner.
The ministry said it would seek to strike a balance between ensuring a stable framework for the remaining oil and gas operations in the North Sea, including jobs and investments, and the country's climate goals.
Only two applications remain in the bidding round, both from the same applicant, Ardent Oil, the Danish Energy Agency said in a statement to Reuters.
All other original applicants, including Lundin Norway, MOL Dania and Total have withdrawn their applications, the agency said.
(Reporting by Nikolaj Skydsgaard; Editing by Barbara Lewis and Elaine Hardcastle)