To investigate the mechanism of synergistic inhibition of oxyresveratrol (OXY) and ascorbic acid (AA) on polyphenol oxidase (PPO), this study systematically evaluated their synergistic effects through the determination of inhibition rates, kinetic analysis, molecular docking, and structural characterization. The results showed that the synergistic treatment of OXY and AA significantly inhibited the browning of fresh-cut potato strips and reduced the activity of PPO, with the most pronounced effect observed at a OXY:AA volume ratio of 2:1. The inhibition rate of PPO increased by 9.08% and 38.12% compared with individual treatments with OXY or AA, respectively. Additionally, the chelation capacity of copper ion was enhanced by 41.07% and 135.45% compared with the individual treatments, respectively. Kinetic analysis revealed that the synergistic inhibition followed a mixed-type mechanism, with inhibition constants for free PPO and enzyme-substrate complexes of 2.46 and 3.27 μmol/L, respectively. Molecular docking results indicated that the OXY-AA-PPO complex formed more hydrogen bonds under synergistic conditions, and the binding energy decreased by 2.32 and 4.45 kcal/mol compared with the individual treatments, respectively, suggesting improved protein conformational stability. Fluorescence spectroscopy and circular dichroism (CD) spectroscopy further demonstrated that the emission fluorescence intensity of PPO treated with the 2:1 combination was 71.93% of that of the original enzyme, and the β-sheet content decreased by 20.2% and 10.4%, respectively, compared with the individual treatments, confirming that the synergistic effect disrupted the tertiary and secondary structures of the enzyme. This study provides a reference for the application of synergistic inhibition technology in the preservation of fresh-cut fruits and vegetables.