Ndi, the leader and founder of the Social Democratic Front (SDF), the main opposition party in parliament, died shortly before midnight on Monday, the party's statement said.
Fru Ndi challenged Biya, who has ruled the West African country with an iron fist for more than 40 years, in the presidential elections of 1992, 2004 and 2011, coming second each time.
The SDF has only five seats in the current parliament. It held 18 in the previous legislature but lost influence to the all-powerful Cameroonian People's Democratic Movement (RDPC) led by 90-year-old Biya, who has ruled Cameroon since 1982.
The SDF is periodically plagued by internal crises and Fru Ndi, whose nickname is "the Chairman," was in recent years contested by a faction of senior party officials.
They accused him, without proof, of enriching himself using public funds, to which all parties represented in parliament are entitled, and of being "bought off" by the ruling party in its attempt to "woo" the opposition.
As he was already ill when the last presidential vote took place in 2018, Fru Ndi encouraged SDF vice-president Joshua Osih to stand.
Osih came fourth, far behind Maurice Kamto, leader of the Movement for the Rebirth of Cameroon (MRC), and third-placed Cabral Libii.
Since then, Kamto, who was imprisoned without trial for nine months in 2019 for peaceful protests against Biya, has become the main opposition figure in Cameroon.
The MRC boycotted the 2018 legislative elections and is not represented in parliament.
Fru Ndi began his political career in the 1980s as a member of Biya's RDPC.
He founded the SDF in 1990 when Cameroon officially ended one-party rule.
The primarily English-speaking Northwest and Southwest regions became part of Cameroon in 1961, and have been plagued by conflict between the army and separatists since the latter declared independence in 2017.
Biya has resisted calls for wider autonomy and responded with a brutal crackdown.
Fru Ndi advocated a federal solution, rather than out-and-out independence for the Anglophone regions, which earned him the wrath of the most radical separatists.
His house was torched and he was kidnapped briefly in 2019 by an armed group, which demanded he pull his deputies out of parliament.
Cameroon Oppo Leader Ndi Dies
DOUALA — John Fru Ndi, a long-time opponent of autocratic Cameroonian President Paul Biya, has died at age 81 after a long illness, his party said on Tuesday.
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