As the main water-soluble active ingredient from Salvia miltiorrhiza, salvianolic acid B (SAB) has a wide range of biological activities. Serum albumin is the most abundant protein in plasma (about 60%) and can bind with various endogenous and exogenous compounds, which can play a role in the storage and transport of compounds. When SAB enters into the human body, it must be combined with the protein in the blood system and then transported to its receptor site and played its pharmacological effects. Thus, in order to better understand the distribution, transport and metabolism of SAB in vivo, the interaction of SAB with bovine serum albumin (BSA) has been investigated using fluorescence spectra, circular dichroism (CD) and nuclear magnetic resonance spectra (NMR) under simulated physiological conditions. The results showed that the binding of SAB to BSA could quench the intrinsic fluorescence of BSA through the combined quenching mechanism (static and dynamic), but the static quenching was the primary one. The binding constant was 7.51×105 L·mol-1 (288 K), 7.40×105 L·mol-1 (298 K) and 5.57×105 L·mol-1 (308 K), respectively. It was found to be in the order of 105 L·mol-1 and decreased with the increasing temperature. The results of site marker competitive experiments indicated that SAB specifically bound to Site I of BSA in the hydrophobic pocket of sub-domain IIA. The stoichiometric ratio between BSA and SAB was calculated by using the Scatchard equation, and the result suggested that the SAB can form a 1∶1 type non-covalent complex with BSA. The three-dimensional fluorescence and CD studies indicated that SAB induced some microenvironmental changes of tryptophan and tyrosine in BSA. That was, the binding of SAB to BSA brought the tryptophan and tyrosine residues to a more hydrophilic environment, while the changes of secondary and tertiary structures of BSA were relatively small. Furthermore, the chemical shift of SAB at various concentrations of BSA was studied using NMR spectra, and the results showed that the benzene ring of H5” and H6” in SAB played a vital role in the binding process during the BSA-SAB complexation. This research will be helpful for understanding the mechanism of SAB in vivo and the influence of the binding of SAB to the conformation and function of serum albumin in biological processes, and can provide some theoretical basis for the development of SAB related new drugs.