Baicalin is a widely used compound in Chinese traditional medicine and exhibits many pharmacological activities. Under simulated physiological conditions, the interaction between baicalin and human serum albumin(HSA), had been investigated by fluorescence spectroscopic, ultraviolet spectrum(UV), Fourier transform infrared(FT-IR), circular dichroism(CD) and molecular modeling. The results indicated that baicalin caused a static quenching of intrinsic fluorescence of HSA. The binding constants were 1.28×105 and 0.91×105 L·mol-1 at 299 and 309 K respectively. The thermodynamic analysis found the enthalpy change (ΔH) and the entropy change (ΔS) were -26.2 kJ·mol-1and 10.1 J·mol-1·K-1 respectively, which suggested that hydrophobic was the predominant forces in the baicalin-HSA complex. The alterations of protein secondary structure in the presence of baicalin in aqueous solution were estimated by the evidences from CD and FT-IR. The results from synchronous fluorescence indicated microenvironment around tryptophan (Trp) had a slight trend of polarity increasing. Molecular modeling suggested baicalin was located in subdomain IIA by hydrophobic forces，which was agreed well with the corresponding experimental results.