Hesperetin-7-O-glucoside (HMG) is a precursor substance of a new sweetener. The enzymatic properties of hesperidinase were determined by high performance liquid chromatography (HPLC). The reaction mechanism and directional regulation of the conversion of hesperidin to HMG were discussed. Results showed that hesperidinase had good stability at 20-70 ℃ and pH 3.0-7.0, the optimum reaction temperature and pH was 60 ℃ and 4.0, respectively. Furthermore, at the optimum reaction temperature and pH, the kinetic constant Km of hesperidinase was 296.69 μg/mL, the maximum reaction rate Vm was 0.872 3 μg/(mL·min). Also, the Km of α -L- rhamnosidase related to HMG transformation was 232.58 μg/mL and the Vm was 0.397 9 μg/(mL·min). The optimal hydrolysis conditions were pH 4.0 and hydrolysis temperature of 60 ℃, enzyme dosage 15.35 U, the amount of solubilizer 0.8%, the amount of glucose 3 mg/mL, and hydrolysis time 3 h. The enzymatic reaction was carried out using these conditions in favor of HMG transformation, and the yield of HMG was 35.8%.