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Tyrosinase is a ubiquitous enzyme that plays an essential role in the production of melanin. Effective inhibitors of tyrosinase have extensive applications in the medical, cosmetic and food industries. In this study, a combination of enzyme kinetics, ultraviolet (UV)-visible absorption, fluorescence spectroscopic techniques and a computational simulation method was used to characterize the inhibitory mechanism of 7,8,4´-trihydroxyflavone on tyrosinase. 7,8,4´-Trihydroxyflavone was found to strongly inhibit the oxidation of l-DOPA by tyrosinase with an IC value of 10.31 ± 0.41 μM. The inhibitory mechanism was determined to be reversible and non-competitive with a K of 9.50 ± 0.40 μM. The UV absorption spectra showed that 7,8,4´-trihydroxyflavone could chelate with copper ions and form a complex with tyrosinase. The intrinsic fluorescence of tyrosinase was quenched by 7,8,4´-trihydroxyflavone through a static quenching mechanism. 7,8,4´-Trihydroxyflavone was found to occupy a single binding site with a binding constant of 7.50 ± 1.20 × 10 M at 298 K. The conformation of tyrosinase changed, and the microenvironment became more hydrophilic after 7,8,4´-trihydroxyflavone binding. Thermodynamics parameters indicated that the binding was a spontaneous process and involved hydrogen bonds and van der Waals forces. The binding distance was evaluated to be 4.54 ± 0.05 nm. Docking simulation analysis further authenticated that 7,8,4´-trihydroxyflavone could form hydrogen bonds with the residues His244 and Met280 within the tyrosinase active site. Our results will contribute to further understanding of the inhibitory mechanisms of 7,8,4´-trihydroxyflavone against tyrosinase and will facilitate future screening for tyrosinase inhibitors.