Tyrosinase is the rate-limiting enzyme controlling the production of melanin, and tyrosinase inhibitors can regulate the overproduction of melanin by inhibiting tyrosinase activity, which is an effective method to treat pigmentation disorders. In this study, kinetic analysis, multispectroscopic methods and molecular simulation were used to investigate the inhibitory activity and mechanism of trilobatin on tyrosinase. The kinetic analysis showed that trilobatin had significant inhibitory activity on tyrosinase in a reversible and mixed-type manner with IC values of (2.24 ± 0.35) × 10 mol L. The intrinsic fluorescence of tyrosinase was quenched by trilobatin through a static quenching mechanism. Different spectroscopic measurements demonstrated that trilobatin could change the microenvironments and conformation of tyrosinase and molecular docking determined the binding site of quercetin on tyrosinase.