Abstract: In order to study the interaction between limonin (LM) and bovine serum albumin (BSA), in this paper, the interaction mechanism between LM and BSA was explored by ultraviolet (UV) spectroscopy, fluorescence spectroscopy and molecular docking technology. The results showed that LM could significantly quench the endogenous fluorescence of BSA, displaying a static quenching type. In addition, LM formed a binding site with BSA by hydrogen bonding and van der Waals forces, demonstrating a spontaneous interaction mechanism. Meanwhile, UV spectroscopy and synchronous fluorescence spectroscopy showed that the interaction between LM and BSA could increase the hydrophobicity of tyrosine (Tyr) and tryptophan (Trp) residue microenvironment on BSA. Through competition site experiments and molecular docking, it was found that the binding site between LM and BSA was near site Ⅰ, and LM could form hydrogen bonds with Trp-213 on BSA, and there were van der Waals forces between LM and Tyr-340/451 residues.