The primary objective of this study is to evaluate the binding characteristics of human serum albumin（HSA） and 5′-hydroxy-2,2′,4,4′,5-pentabromo diphenyl ether（5′-OH-BDE-99） in physiological conditions.Several spectroscopy techniques such as steady-state fluorescence spectroscopy,time-resolved fluorescence,FTIR and three-dimensional fluorescence spectrometry, combined with molecular docking and molecular dynamics simulation were employed to comprehensively analyze the mechanism of interaction between 5′-OH-BDE-99 and HSA.The results of fluorescence spectroscopy experiments,non-radiation energy transfer theory and dynamic simulation showed that 5′-OH-BDE-99 could quench the intrinsic fluorescence of HSA through static quenching mechanism.The experiments of FTIR,three-dimensional fluorescence spectra and dynamic simulation revealed that the binding of 5′-OH-BDE-99 to HSA could induce conformational and microenvironmental changes.Moreover,according to molecular docking and thermodynamic method,hydrophobic force is the main acting force.