Horseradish Peroxidase (HRP) was immobilized on layered hydrogen peroxide, which was synthesized by hydrothermal method. Catalytic degradation characteristics of phenol wastewater by immobilized enzyme was studied under the conditions of microwave and ultrasonic combined treatment. Immobilized enzyme catalytic activity was investigated by means of UV analysis and kinetic constants of HRP immobilization were determined by Lineweaver-Burk double-reciprocal plot method. A comparison to phenol degradation efficiency and storage stability of immobilized enzyme under different conditions was conducted with liquid chromatography method. The results showed that the degradation efficiency of the immobilized enzyme was 74.4% under microwave-ultrasonic irradiation conditions, which was increased by 43.6% when phenol concentration was 60 mmol/L. Microwave–ultrasonic irradiation enhances enzyme activity, and hence the catalytic efficiency, by accelerating the decomposition of the said inhibitors. It was also found that the acceleration effect strengthens with an increased phenol concentration.After continuous use seven times, the degradation efficiency of irradiation group is still 5.06 times of that of the control group.