<p>Based on the crucial role of tyrosinase (TYR) in pigment metabolism, food browning, and disease regulation, as well as the urgent need for the development of its inhibitors, in this study, a three-dimensional quantitative structure–activity relationship (3D-QSAR) pharmacophore model was constructed to screen the database and obtain potential TYR inhibitors. The inhibition mechanism of the potential TYR inhibitors—sophoricoside (Sop) and puerarin (Pue)—on TYR was explored by enzyme kinetics, multi-spectral technology, and molecular simulation technology. The results showed that both Sop and Pue exhibited reversible mixed-type inhibition of TYR. The half maximal inhibitory concentration (IC₅₀) values determined through the TYR activity experiment were 0.45 ± 0.02 μM and 1.54 ± 0.07 μM, respectively. Fluorescence spectroscopy analysis indicated that their binding to TYR belonged to the static quenching mechanism, and complexes were formed through electrostatic interactions, hydrogen bonds, and hydrophobic interactions at a molar ratio of 1:1 with TYR. Fourier transform infrared spectroscopy, molecular docking, and molecular dynamics simulations showed that Sop disrupted the hydrogen bond network structure near the α-helix region of TYR, while Pue promoted the formation of the α-helix and reduced the content of random coils, making the structure of TYR more compact. These research results suggest that Sop and Pue alter the conformation of TYR and inhibit its activity, which provides a theoretical basis for their potential applications in fields such as cosmetics, medicine, and food preservation.</p>