Abstract Polyphenol oxidase is the popular enzyme involved in fruit-vegetable browning and melanin synthesis. In the present paper, L-3-phenyllactic acid, a natural bacteriostatic substance, was investigated as an inhibitor of polyphenol oxidase. The results were demonstrated that the residual enzyme activity of polyphenol oxidase decreased gradually with the increase of the concentration of L-3-phenyllactic acid. The L and ΔE values of the reaction system increased gradually. The reversible mixed-type inhibition mode of L-3-phenyllactic acid was determined by Lineweaver Burk plot. At the same time, the results of fluorescence quenching demonstrated that L-3-phenyllactic acid was a quencher of polyphenol oxidase, and the molecular docking study provided the binding mode of L-3-phenyllactic acid and polyphenol oxidase at the molecular level. L-3-phenyllactic acid decreased the activity of polyphenol oxidase and browning of fresh-cut Agaricus Bosporus. This research first studied the inhibitory effect of L-3-phenyllactic acid on the activity of polyphenol oxidase, and would provide a theoretical foundation for the use of L-3-phenyllactic acid as anti-polyphenol oxidase agents.