We produced single-chain recombinant gonadotropins (rGTHs), follicle-stimulating hormone (rFSH) and luteinizing hormone (rLH) of goldfish Carassius auratus, in which the FSHβ or LHβ subunit was fused to the α subunit, by baculovirus in silkworm larvae Bombyx mori. Biological activities of rGTHs were confirmed in vivo by using male goldfish, female bitterling Rhodeus ocellatus ocellatus, and male Japanese eel Anguilla japonica. Administration of rFSH or rLH induced milt production in male goldfish. rLH induced ovulation in bitterling but rFSH showed lower potency. Both rLH and rFSH induced spermatogenesis in male eels after 6 administrations of rGTHs at seven days interval. Spermatozoa were observed in rLH-administered fish. It is suggested that the single-chain goldfish GTHs could be used for the induction of gonadal development in aquaculture fishes.