In this paper, the binding of flavonoids (quercetin and rutin) to hemoglobin (Hb) have been investigated by fluorescence, absorption spectroscopy and circular dichroism (CD) spectroscopy. The binding parameters and binding mode between flavonoids and Hb are determined and the results of CD and synchronous fluorescence spectra indicate a conformational change of Hb with addition of flavonoids. The effects of lecithin liposomes on the binding parameter of quercetin and rutin to Hb are also studied. When incorporated into liposome, flavonoids can reduce the fluorescence of tryptophanyl residues of Hb to a lesser extent. The difference of the structure characteristics between quercetin and rutin has a significant effect on their binding affinity for Hb.