The invention provides a preparation method of Cu2MoS4 nano-enzyme, and the method comprises the following steps: mixing cuprous oxide, an organic carbon source, an inorganic molybdenum source and anorganic sulfur source in an organic solvent, and carrying out high-temperature calcination to obtain the hollow Cu2MoS4 nano-enzyme. Compared with the prior art, the Cu2MoS4 nano-enzyme prepared by the invention has catalase activity and greatly relieves tumor hypoxia, and meanwhile, the nano-enzyme also has peroxidase activity and can consume glutathione overexpressed in a tumor, so that an antioxidant defense system of the tumor is destroyed, and the chemokinetic treatment efficiency is enhanced; two pairs of redox couples exist, the redox couples can react with hydrogen peroxide to generatehigh-toxicity hydroxyl radicals, a remarkable chemical and dynamic curative effect is achieved, meanwhile, the nano-enzyme has strong absorption in the whole near-infrared area, and photo-thermal therapy can be achieved; and under laser excitation, superoxide anion free radicals can be generated and used for photodynamic therapy.