Abstract: To investigate the effect of phenolic acids on the activity of β-glucosidase, this study systematically evaluated the activation mechanism of p-hydroxybenzoic acid (PHBA) at varying concentrations (0~0.5g/L) through circular dichroism spectroscopy and molecular docking, with a focus on enzyme conformational dynamics and molecular interactions. The results showed that PHBA significantly enhanced β-glucosidase activity in a concentration-dependent manner. Secondary structural analysis revealed that the content of α-helix significantly decreased by 52.14% (P<0.05), while β-sheet, β-turn, and random coil structures increased by 90.20%, 27.24%, and 50.56%, respectively, at 0.5 g/L PHBA compared to the control group. These structural shifts suggestted that the enzyme’s active site might reside within α-helix regions. Fluorescence quenching assays confirmed spontaneous binding between PHBA and β-glucosidase, with one or one class of binding sites identified. Molecular interaction analyses revealed that hydrogen bonding, van der Waals forces, and hydrophobic interactions predominantly stabilized the PHBA-enzyme complex. Molecular dynamics simulations further validated the structural stability of the PHBA-β-glucosidase complex over 100 ns trajectories (RMSD<0.4 nm), confirming PHBA as a potent β-glucosidase activator. This study elucidates the molecular basis of PHBA-driven enzyme activation and provides a theoretical foundation for enhancing β-glucosidase activity in phenolic acid-rich fermented fruit and vegetable products.