The binding interactions of gold nanoparticles with trypsin were investigated using multi-spectra methods and molecular modeling. The experiment data showed that trypsin modified the surface of gold nanoparticles. The fluorescence intensity of trypsin was quenched by gold nanoparticles that strongly associated with protein and induced the inhibition of enzyme activity. The electrostatic and hydrophobic interactions were the primary contributors to the binding forces between trypsin and gold nanoparticles. The covalent interactions might be also involved in the binding process. The modeling calculated results indicated that the binding site was near to the primary substrate-binding pocket and the active site of the enzyme substrate. This work elucidated the interaction mechanism of trypsin with gold nanoparticles from the theoretical and experimental angle.