Herein, the protein binding rates of structurally different flavonoids to human serum albumin (HSA) were elucidated by applying the high performance affinity chromatography (HPAC). The flavonoids with hydroxyl groups on ring A showed a higher protein binding rate compared with those that there was no hydroxyl on ring A. However, the hydroxylation of ring B lowered the protein binding rate. It was also found that an additional methoxy group in flavone ring A would decrease the protein binding rate. Nevertheless, the methoxy group in flavanone ring A (position 6) and isoflavone ring B (position 4′) increased the protein binding rate. Methoxy group at other positions of flavonoids slightly enhanced or no significantly affected the binding rates on human serum albumin. Hydrogenation of C2[dbnd]C3 double bond of flavonoids decreased the protein binding rate and had the same effect as glycosylation which decrease the protein binding rate by 5%–25%.