Abstract：NMN (nicotinamide mononucleotide) transferase and ethanol dehydrogenase were covalently immobilized with magnetic nanoparticles; to produce NADH (nicotinamide adenine dinucleotide). The conditions of co-immobilization, the properties and kinetics of co-immobilized enzyme were investigated. The optimized immobilization condition as follow : NMN transferase and alcohol dehydrogenase are 6.5 U/0.5 mg and 10.3 U/0.5 mg, respectively, and the system pH is controlled at 5.0-7.0 to immobilize the enymes for 2 h at 25℃, under which condition, NADH yield could reach 87%. After 11 reuses of the co-immobilized double enzyme, the residual enzyme activity still remains at about 61.1%, which showed a good operational stabilization. The deduction and validation of the kinetic equation of two enzymes showed that the reaction system of co-immobilized double enzymes accorded with Michaelis equation, and its kinetic reaction rate depended on the reaction rate of immobilized ethanol dehydrogenase.