Comparative molecular field analysis (CoMFA) techniques were used to perform three-dimensional quantitative structure-activity relationship (3D-QSAR) studies on the anti-tumor activity (pI_H and pI_C) of 28 fluoroquinolon-3-yl s-triazole sulfide-ketone derivatives (FQTSDs) against two cancer cell lines, including human hepatoma Hep-3B cells and human pancreatic cancer Capan-1 cells. 23 compounds were randomly selected as the training set to establish the prediction models, which were verified by the test set of 6 compounds containing template molecule. The obtained cross-validation (R_cv²) and non-cross-validation correlation coefficients (R²) of the CoMFA models were 0.477 and 0.850 for pI_H, and 0.421 and 0.836 for pI_C, respectively. The contributions of steric and electrostatic fields to pI_H were determined to be 48.1% and 51.9%, and those to pI_C were 49.4% and 50.6%, respectively. The CoMFA models were then used to predict the activities of the compounds in the training and testing sets, and the models had a strong stability and good predictability. Based on the 3D contour maps, four novel FQTSDs with a higher anti-tumor activity were designed. However, the effectiveness of these novel FQTSDs is still needed to be verified by experimental results.