The present invention provides a novel process for preparing nicotinamide adenine dinucleotide phosphate (NADP). The process of the present invention comprises performing phosphorylation using a polyphosphoric acid or a salt thereof and nicotinamide adenine dinucleotide (NAD+) as substrates in the presence of a polyphosphate-dependent NAD+ kinase from a Mycobacterium, wherein the reaction solution contains 0.1-15% by weight of the polyphosphoric acid or a salt thereof, and 5-150 mM of a divalent metal ion.