OBJECTIVE： To study the interaction mechanism between flavonoids and human serum albumin （HSA）， and to compare the effects of different B-ring substitutions （hydroxyl， methoxyl group） of flavonoids on macromolecular receptor. METHODS： The interaction regularity between three flavonoids with different B-ring substitutions （quercetin， hesperetin， methyl hesperetin） and HSA was studied with fluorescence spectroscopy， the fluorescence quenching types between 3 flavonoids and HSA were determined and analyzed， and the velated binding constant， binding site and thermodynamic parameters were calculated. RESULTS： The quenching constants （Ksv） and binding constants （KA） were decreased with the increase of temperatures. The number of binding site （n） was approximately equal to one， and the thermodynamic parameters ΔH＜0，ΔS＞0， the binding interaction of these compounds with macromolecules was influenced because of the difference of the B-ring substituents. CONCLUSIONS： The quenching mechanism between three flavonoids and HSA was static quenching； the number of binding site was one； the interaction force of the three compounds with HSA was mainly static electricity， and hydroxyl group in the B-ring was likely the major active group and exerted stronger binding force than methoxyl group to connect with macromolecules.