Members of the Biafran Zionist Movement declared independence, raised the Biafran flag and then marched through the region's main town, Enugu.
More than one million people died during the 1967-70 Biafran conflict - mostly from hunger and disease.
Most of those arrested were young men but some were veterans of the war.
They were all remanded in custody.
Biafran War 1967-1970
1960: Nigeria gains independence from the UK
1967: South-eastern portion of Nigeria secedes as Republic of Biafra on 30 May
Biafra dominated by Igbo ethnic group
Home to much of Nigeria's oil
Nigerian army blockades Biafra and more than a million people die through famine, disease and fighting
1970: Biafran government surrenders
The BZM first gathered on Sunday to mark the birthday of former Biafran leader Chukwuemeka Ojukwu, who died in November 2011 and was buried in March.
The BBC's Will Ross in Lagos says that 45 years after the Biafran flag was first raised - an action which sparked Nigeria's civil war - a small number of separatists still keep their dream alive, despite the threat of being charged with treason.
Our correspondent says the war has been put back in the spotlight as the renowned Nigerian writer Chinua Achebe has just released his memoirs of the conflict.
He also notes that as the novel Half of a Yellow Sun by the author Chimamanda Ngozi Adichie is being made into a film, this traumatic period of Nigeria's history is set to reach a wider audience.
The Biafran leaders were mostly from the Igbo ethnic group, whose members were targeted by rioters in northern areas following a 1966 coup seen as being led by Igbo officers.
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