The in vitro anti-proliferative activity (pIC_i, i = hp, ca, hl) of fluoroquinolone (rhodanine α,β-unsaturated ketone) amide compounds, referred to as “fluoroquinolone amide derivatives (FQADs)” towards Hep-3B, Capan-1 and HL60 cells, was studied by the 3D-QSAR method of comparative molecular field analysis (CoMFA). Based on the training set of 14 compounds, the prediction model was established, which was further verified by the test set of 5 compounds with template molecule included. It is found that steric and electrostatic fields contribute 66.8% and 33.2% to pIC_hp, 61.4% and 38.6% to pIC_ca, and 61.5% and 38.5% to pIC_hl, respectively. The R_cv² (i.e, cross-validation coefficient) is 0.324, 0.381, and 0.421 for pIC_hp, pIC_ca, and pIC_hl, respectively, while the corresponding R² (i.e, non-cross-validation coefficient) all reach 0.999. Then, the models were employed to estimate the activities of the training and test compounds, and the results show that the stability and predictability of developed models are very satisfactory. According to the contour maps of steric and electronic fields, bulky groups linked to 2-, 3-, 4-positions of phenyl ring, and electropositive groups near the 4-position and electronegative groups far away may increase the anti-proliferative activity. Using the information provided by the 3D contour maps, four new FQADs owing higher anti-     proliferative activity were designed, but their effectiveness should be further tested by experiments.