The interaction between the flavonoid hesperidin and bovine serum albumin (BSA) was investigated by fluorescence and UV/Vis absorption spectroscopy. The results revealed that hesperidin caused the fluorescence quenching of BSA through a static quenching procedure. The hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The binding site number n, and apparent binding constant K A , corresponding thermodynamic parameters ΔG o , ΔH o , ΔS o at different temperatures were calculated. The distance r between donor (BSA) and acceptor (hesperidin) was obtained according to fluorescence resonance energy transfer. The effect of Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ , and Mn 2+ on the binding constants between hesperidin and BSA were studied. The effect of hesperidin on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy and UV/Vis absorption spectroscopy.