The binding reaction of rutin-Sm with serum albumin (SA) was investigated by the fluorescence method in physiological condition. The authors studied mainly the quenching mechanism of the fluorescence of SA by rutin-Sm, and calculation of the binding constants K(LB) of human serum albumin (HSA) and bovine serum albumin (BSA) with rutin-Sm by Lineweaver-Burk equation at different temperatures respectively, then obtained the thermodynamic parameters of HSA and BSA with rutin-Sm according to the calculated binding constants K(LB) at different temperature, meanwhile the type of binding forces of HSA and BSA with rutin-Sm was determined. The results showed that the emission spectra of BSA (HSA) in the presence and absence of rutin-Sm are different. The emission spectra of BSA (HSA) in the presence of rutin-Sm can be quenched. The quenching mechanism of rutin-Sm to SA was static quenching with non-radiation energy transfer for new complex of SA and rutin-Sm. The binding constants K(LB) (L x moL(-1)) were 6.540 x 10(5) and 3.265 x 10(5) for BSA, and 6.830 x 10(5) and 4.665 x 10(5) for HSA at 295 K and 310 K respectively. And the type of bonding forces was estimated by the calculation of thermodynamic parameters of the reaction of rutin-Sm with SA at different temperatures, and the result showed that the binding forces were mainly H-bond and Van der Waals between BSA and rutin-Sm due to the deltaH < 0 and deltaS < 0, and the main electrostatic interaction of rutin-Sm and HSA because of deltaH < 0 and deltaS > 0. The effect of rutin-Sm on the conformation of serum albumin was also studied by using synchronous fluorescence spectroscopy. Results indicated that rutin-Sm could be deposited and transported by serum protein in vivo.