The National Council of Resistance of Iran, which first made allegations of the tunnels in September, said their sources in Iran had evidence of underground complexes in 14 locations, near Tehran, Isfahan, Qom and other cities.
"These have been built by military agencies and their front companies," said Hossein Abedini, a member of the foreign affairs committee of the NCRI at a news conference.
"The purpose of the tunnels is to conceal parts of the Tehran regime’s atomic and missile programmes," he said, adding they were used for hiding research centres, workshops, nuclear equipment and nuclear and missile command and control centres.
He said the NCRI, which has previously reported accurately about hidden atomic facilities in Iran, had sent the Vienna-based International Atomic Energy Agency its latest information and urged the body to inspect the tunnels.
"Today we call on the International Atomic Energy Agency to immediately and urgently act," said Abedini.
No one was immediately available at the IAEA to comment.
The NCRI is a coalition of exiled opposition groups that seeks to overthrow Iran’s clerical rulers and is listed by the United States as a terrorist organization.
The allegations come a day before Britain, France and Germany meet Iranian officials to discuss the possibility of restarting negotiations on Iran’s disputed nuclear programme.
Almost two years of talks collapsed earlier this year when Iran said it would restart some sensitive nuclear activity. The West suspects Iran of developing atomic weapons but Tehran argues it needs nuclear technology to generate electricity.
Abedini gave more details about the tunnels than previously available and named companies and engineers involved.
He said a complex of tunnels at Parchin, east of Tehran, was used for work on laser techniques for uranium enrichment.
Another tunnel system in Khojeer, southeast of Tehran, was used to assemble ballistic missiles and was 1 km (0.6 miles) long and 12 metres (yards) wide.
The NCRI believes Iran could develop an atomic bomb in "a couple of years".