Gelatinase A (MMP-2) has been studied and proven to play a vital role in the intrusion and metastasis of cancer. Flavonoids influence on molecular and cellular functions of MMP-2 and thus a systematic investigation of flavonoids against the metalloproteolytic activity of MMP-2 has been performed in this study. A fluorescence resonance energy transfer method was used to investigate the inhibitory activities of various flavonoids. Flavone, flavonol and isobavachalcone derivatives showed their inhibitory activity against MMP-2. Surprisingly, the most effective inhibitor was Amentoflavone and its blocking function was superior to other flavonoids. Its IC50 value was 0.689 μM. An induced-fit docking study was carried out to survey its extraordinary activity. The binding mode of Amentoflavone is quite similar to that of (2 ∼ {S})-2-[2-[4-(4-methoxyphenyl) phenyl] sulfanylphenyl] pentanedioic acid complexed with MMP-9. Amentoflavone interacts with the functional zinc and catalytic residue, Glu202. Therefore, the docking study reasonably confirmed the strong inhibitory activity of Amentoflavone.