Abstract: To discover an efficient optimization method for modification of the fluoroquinolone C-3 carboxylic group, the title fluoroquinolon-3-yl s-triazole-acylhydrazone methylsulfide derivatives( 6a - 6q )were designed and synthesized from pefloxacin on the basis of pharmacophore combination principle with a functionalized acylhydrazone group as a modified side-chain for the C-3 bioisosteric s-triazole of pefloxacin. The structures were characte-rized by element analysis and spectral data, and the in vitro antitumor activity against SMMC-7721, L1210 and HL60 cell lines was evaluated by MTT assay. The results showed that the title compounds exhibited more significant inhibitory activity than that of the parent pefloxacin, in which compounds with electron-withdrawing group attached on aryl ring had more potency than that of compounds with electron-donating group, especially compounds with carboxylic substituent were comparable to control doxorubicin. It suggests that carboxylic residue around the C-3 bioisostere is favorable to improve the antitumor activity.