The interaction mechanism between trypsin and fulvic acid was analyzed by multispectral method and molecular docking simulation. The fluorescence spectra showed that fulvic acid induced static quenching of trypsin. The validity of this conclusion was further substantiated through the computation of the binding constants. The thermodynamic parameters show that the reaction is mainly controlled by van der Waals force and hydrogen bond force, and the reaction is spontaneous. In addition, based on the obtained binding distance, there may be a non‐radiative energy transfer between the two. The ultraviolet spectrum showed that fulvic acid could shift the absorption peak of trypsin, indicating that fulvic acid had an effect on the secondary structure of trypsin. According to the synchronous fluorescence spectrum results, fulvic acid primarily interacts with tryptophan residues in trypsin and induces alterations in their microenvironment. Three‐dimensional fluorescence spectrum and circular dichroism further proves this conclusion. The molecular docking simulation reveals that the interaction between the two groups primarily arises from hydrogen bonding and van der Waals forces. The findings suggest that FA has the ability to induce conformational changes in trypsin's secondary structure.