The interaction between dihydromyricetin (DMY) with human serum albumin (HSA) under the physiological conditions was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra and UV-visible absorption spectroscopy. In the mechanism discussion it was proved that the fluorescence quenching of HSA by DMY is a result of the formation of DMY-HSA complex. Binding parameters calculated showed that DMY bind to HSA with the binding affinities of the order 105∼106 L�mol-1. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -28.76 kJ�mol-1 and 18.21 J�mol-1�K-1, respectively, which implied that the hydrophobic and hydrogen bonds interactions play predominant roles in the binding process. The binding site of DMY on HSA may be located in hydrophobic cavity of subdomain IIA by the analysis data of fluorescence and synchronous fluorescence spectra. The specific binding distance r (3.37 nm) between donor (Trp-214) and acceptor (DMY) was obtained according to Forster non-radiative resonance energy transfer theory. CD spectral result demonstrates that DMY does not affect the secondary structure of HSA and can maintain protein stabilization. In addition, the effect of some common metal ions (e.g. Zn2+, Cu2+, Co2+, Ni2+, Fe3+) on the binding constant between DMY and HSA was examined.