The molecular mechanism of the interaction between resveratrol and trypsin was studied using fluorescence spectroscopy (intrinsic fluorescence, synchronous fluorescence, three-dimensional fluorescence), ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and the molecular docking method, as well as through enzyme activity and antioxidant assay. The fluorescence experiments (the Stern–Volmer quenching constants (K_SV)) indicated that resveratrol quenched the intrinsic fluorescence of trypsin through the static quenching mechanism. The number of binding sites was about one. The thermodynamic functions ΔG < 0 and ΔH > 0, ΔS > 0 of the binding process, indicating that the combination of the resveratrol and trypsin processes was a spontaneous exothermic reaction and that the hydrophobic effect was the main force between them. UV-vis spectra, synchronous fluorescence spectra and three-dimensional fluorescence spectra analysis showed that the combination of resveratrol and trypsin induced changes in the microenvironment around the fluorophores of trypsin, resulting in alterations in the spatial structure of trypsin. FT-IR spectroscopy indicated that the contents of the α-helixes and β-turns in trypsin decreased and that the contents of the β-sheets, random coils and antiparallel β-sheets increased. All these experimental results were verified and reasonably explained by the molecular docking results. Upon resveratrol and trypsin binding, the enzyme catalytic activity of trypsin and the antioxidant of resveratrol decreased. Results from this study would be useful in elucidating the molecular mechanisms of the interactions between resveratrol and trypsin and contribute to making full use of resveratrol in the food industry.