Hakainde Hichilema, president of the United Party for National Development (UPND), and his deputy Geoffrey Mwamba were arrested on suspicion of sedition, police in the copper-producing southern African state said.
Sedition is a crime in Zambia defined by conduct inciting people to rebel against the authority of the state, and carries a maximum penalty of seven years in prison.
"They have been arrested and detained for seditious practices and unlawful assembly which occurred on Sept. 26. They will appear in court tomorrow," Copperbelt police chief Charity Katanga told Reuters.
Hichilema continues to dispute the August election outcome and in a statement before his arrest compared the government's conduct to the limitation of freedoms experienced when Zambia was still a British colony.
"...We are telling Lungu and his disputed regime that we shall not stop moving around the country to meet our structures and greet our people," Hichilema said.
Katanga said another 61 opposition supporters were arrested after they threw stones at police following the arrest of the two UPND leaders.
Last month the Lusaka High Court threw out an attempt by Hichilema to overturn a Constitutional Court decision not to give him more time to legally challenge Lungu's re-election.
Lungu said last month that Zambians should put the divisive elections behind them and work as one to develop the country.
He said after being sworn in for a new five-year term that he would focus on unlocking agricultural potential in Zambia to reduce its dependence on copper mining.