Daphnetin (7,8-dihydroxycoumarin), one of the major bioactive components isolated from Daphne odora (as glucoside), is a chelator, an antioxidant and a protein kinase inhibitor. In this paper, we report for the first time studies on the binding of daphnetin to bovine serum albumin (BSA) under physiological conditions with BSA concentration of 1.5 x 10(-6) mol l(-1) and drug concentration in the range of 6.7 x 10(-6) to 2.0 x 10(-5) mol l(-1). Fluorescence quenching spectra in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to investigate the drug-binding mode, the binding constant and the protein structure changes in the presence of daphnetin in aqueous solution. The curvature of Scatchard plot indicates that daphnetin binds to more than one class of sites on BSA. The thermodynamic parameters, enthalpy change (deltaH) and entropy change (deltaS) were calculated to be -24.21 kJ mol(-1) and 19.30 J mol(-1) K(-1) according to van't Hoff equation, which indicated that hydrophobic and electrostatic interaction played main role in the binding of daphnetin to BSA.