In order to clarify the interaction mechanism of the formation of the resveratrol-hordein nanoparticle, the fluorescence, UV-Vis spectroscopic, FTIR and DSCwere used to study the binding reaction between resveratrol and hordein. The fluorescent emission of hordein was inhibited by resveratrol in a dose dependent manner. Fluorescence spectroscopy and DSC indicated that resveratrol interacted with hordein and formed a new complex by a static process. The binding constant(KA(298 K)=2.21×105 L·mol-1, KA(310 K)=1.53×104 L·mol-1) and the number of binding sites(n298 K=1.23, n310 K=0.94)were calculated based on the quenching effect of resveratrol on hordein. Thermodynamic parameter and FTIR indicated that the interaction force between resveratrol and hordein was mainly hydrogen binding and van der Waals force. The binding distance(r0=3.25 nm)between resveratrol and hordein and the energy transfer efficiency (E=0.227) were obtained according to non-radiative energy transfer theory. The effect of resveratrol on the conformation of hordein was further analyzed by using synchronous fluorescence spectrometry. The results indicated that resveratrol changed the hydrophobicity of tryptophan residue, which caused an obvious influence on the conformation of hordein.