Spectroscopic investigation on the interaction of 3,7-dihydroxyflavone with different isomers of human serum albumin

► A novel method for determining the drug binding site on protein has been developed. ► Fluorescence quenching mechanism of HSA has been clarified with multiple approaches. ► Structural alterations of 3,7-diHF after binding to protein has been illustrated. ► Spectroscopic features of 3,7-diHF after binding to protein has been clarified. ► Binding mode of 3,7-diHF and HSA has been investigated and discussed.