The interaction between xanthan gum and trypsin was studied using fluorescence spectroscopy, UV-vis absorption spectroscopy, circular dichroism chromatography, synchronous fluorescence spectroscopy, and molecular docking simulation. The quenching constant (KSV) and dynamic fluorescence quenching rate constant (Kq) were calculated in the study of fluorescence spectra, confirming that trypsin on xanthan gum exhibits static quenching. The interaction distance between xanthan gum and trypsin (r = 2.350 nm) is calculated using Förster theory, indicating non-radiative energy transfer occurs between them. Based on the formula, ΔH, ΔS, ΔG (298K, 303K, 308K) are all negative. Therefore, it can be concluded that the interaction force between xanthan gum and trypsin mainly consists of hydrogen bonds and van der Waals forces. The UV-vis absorption spectra were used to investigate the interaction between the two compounds. It was observed that the absorption peak of trypsin shifted towards longer wavelengths, indicating a change in its conformation. The addition of xanthan gum altered the environment and conformation of tryptophan residues in trypsin molecules, as shown by synchronous fluorescence spectra. The molecular docking simulation method shows that the main forces of xanthan gum and TRY are hydrogen bond and van der Waals force. The findings align with the thermodynamic parameters, suggesting that xanthan gum alters the secondary structure of trypsin. In the three-dimensional fluorescence spectrum, a redshift is observed in the maximum emission wavelengths of PEAK 1 and PEAK 2, indicating structural changes in trypsin upon binding with xanthan gum. Circular dichroic chromatography results demonstrate a significant decrease in β-fold content, implying an effect of xanthan gum on trypsin's structure.