The use of oxidoreductases as biocatalysts for industrial production of valuable compounds has a strong demand for NADP. Herein, we prepared superparamagnetic NAD kinase catalyst to synthesize NADP in vitro. First, Fe3O4 particles were synthesized through a solvothermal method, followed by the chemical modification with epichlorohydrin, iminodiacetic acid, and Ni2+ to yield functional Fe3O4 sub-microspheres. Subsequently, NAD kinase of Escherichia coli was overexpressed and immobilized on to the surface of magnetic sub-microspheres. The immobilized NAD kinase was used to catalyze the conversion of NAD to NADP in a cell-free system. Under optimal condition, the conversion ratio of NAD reached 91.7% and remained at 86.3% after repeated use for five times. Our study revealed that the novel magnetic NAD kinase catalyst possessed favorable properties for magnetic manipulation and NADP production.