Cathepsin B (CatB) is a member of the papain superfamily of cysteine proteases and has been implicated in the pathology of numerous diseases, including arthritis and cancer. Amentoflavone is found in a number of plants with medicinal properties, including Ginkgo biloba and Hypericum perforatum (St. John's Wort). Herein, we report the structure-activity relationship (SAR) and binding mechanism of three biflavones, amentoflavone (AMF1), 4'''-methylamentoflavone (AMF2) and 7'',4'''-dimethylamentoflavone (AMF3), isolated from Taxodium mucronatum by us as novel natural inhibitors of human CatB with strong inhibitory activities at IC50 values of 1.75, 1.68 and 0.55muM, respectively. Density functional theory (DFT) method was applied to optimize the geometry structures of AMF1, AMF2 and AMF3 at the B3LYP/6-31G* level. FlexX was explored to dock the three biflavones to the binding sites of CatB, and to get a better understanding of vital interactions between these biflavones and CatB. A good correlation between the calculated quantum descriptors and the experimental inhibitory activities suggested that quantum model of these potential inhibitors is reliable. Through geometry and electron structure analysis of AMFs, it was observed that the CH3 substitute at 7'' and 4''' positions could not vary the difference in geometry structure significantly, but increase the electron density of A-ring, HOMO energy, hydrophobic property, and improve inhibitory activity. Structural and energetic analysis of AMFs and AMFs-CatB complexes showed that the electron-donor site is the A-ring, which shows the highest HOMO energy distribution, and the electron-acceptor site is the F-ring, which shows the highest LUMO energy distribution in AMFs, and the pi-pi interaction between A-ring and residue Trp221, two hydrogen bonds (O5 and Trp221; O4 and Gln23 ), hydrophobic interaction between the C-ring and residue Cys29 and CH3 substitutes at 7'' and 4''' might play a crucial role in the inhibition of AMFs on CatB. Results indicated that AMFs are new natural reversible inhibitors that would be useful in developing potent inhibitors of CatB.