Abstract: To develop a novel purine degrading enzyme that can be used in the food industry, this study aimed to screen an enzyme with potential purine degradation activity using inverse virtual screening technology and molecular docking technology, clone express, purify, and validate the activity of this enzyme, and explore the optimal reaction conditions for its degradation of purines. The results showed that the docking binding energies of dihydroorotase screened by the inverse virtual screening platform PharmMapper with adenine, guanine, hypoxanthine, and xanthine were −7.3, −6.9, −7.6, and −8.8 kcal/mol, respectively, which had the optimum docking result. The dihydroorotase with a molecular weight of 41 kDa was obtained with cloning expression. The optimal conditions for catalyzing the degradation of guanine were pH8.0, 25 °C, and under the catalysis of Zn²⁺ (0.5 mmol/L), guanine could be degraded at a ratio of 46.1%. Therefore, it found that dihydroorotase had the degradation activity for guanine, and the optimal reaction conditions for guanine degradation were determined, which laid foundation for the application in degradation of food purine.