Abstract: To improve the antitumor activity of fluoroquinolones for a promising development of druggability, twelve novel fluoroquinolone C-3 s-triazole sulfide-one thiosemicarbazone derivatives( 6a - 6l )were designed and synthesized with a functionalized sulfide-one thiosemicarbazone as a modified side-chain for the C-3 bioisteric s-triazole ring of pefloxacin( 1 ). The structures were characterized by elemental analysis and spectral data。The in vitro antitumor activity of novel compounds against SMMC-7721, L1210 and HL60 cell lines was evaluated. The preliminary pharmacological results demonstrated that the title compounds exhibited more significantly antiproliferative activity than either the parent 1 or the corresponding sulfide-one intermediates( 5a - 5l ). In particular, compounds bearing a hydroxyl group or a fluorine atom attached to benzene ring were comparable to the control doxorubicin with an IC₅₀ value of micro-molar concentration, respectively. It suggests that an azole ring modified with functional side-chain instead of the C-3 carboxylic group is favorable to the improve ment of antitumor activity.