<p>Pepsin is a crucial digestive enzyme in the human body. However, its hyperactivity can readily contribute to the development of gastrointestinal disorders. Research has indicated that certain phenolic acids can bind to pepsin, thereby attenuating its activity, which may be affected by the molecular weight and conformation of phenolic acids. Nonetheless, there are few reports in the literature regarding comparative studies on the inhibition of pepsin by various phenolic acids. Hence, three phenolic acids—ferulic acid, chlorogenic acid, and gallic acid—were selected for investigation in this study. A combination of spectroscopic experiments, molecular docking simulations, and antioxidant tests were employed to elucidate the interactions and mechanisms between these phenolic acids and pepsin. The findings demonstrated that the interaction between the three phenolic acids and pepsin occurred via a static quenching mechanism. Notably, chlorogenic acid exhibited the most pronounced impact on the fluorescence of pepsin. At temperatures of 298&#xa0;K and 310&#xa0;K, the fluorescence intensity of pepsin was reduced by 75.23% and 76.17% at a concentration of 0.6 × 10⁻³ mol/L, respectively. Furthermore, thermodynamic parameter analysis indicated that the interaction between the polyphenols and pepsin was a spontaneous process, primarily driven by van der Waals forces and hydrogen bonding interactions. Molecular docking results corroborated the thermodynamic analysis, highlighting hydrogen bonding as the predominant force. Through an analysis of binding free energy, it was determined that the binding affinity of phenolic acids to pepsin follows the order: chlorogenic acid &gt; gallic acid &gt; ferulic acid. Furthermore, upon interaction with pepsin, the DPPH radical scavenging activity of these phenolic acids was diminished. The findings of this study may enhance the understanding of the health implications of phenolic acids in future research.</p>