A nucleotide production process comprises: decomposing an RNA by using a nuclease P1 so as to obtain nucleotides AMP, GMP, CMP and UMP, converting part or all of the nucleotide AMP into a nucleotide IMP by using adenosine deaminase, separating the obtained nucleotide by using an ion exchange resin, and then performing concentration and crystallization to obtain purified nucleotides AMP, GMP, CMP, UMP and IMP or obtain purified nucleotides GMP, CMP, UMP and IMP. The whole biocatalysis production of nucleotides is implemented by using a double-enzyme catalysis method, and high-purity nucleotides are obtained by using an ion resin separation technology and a solvent crystallization method; and the production process is simple and environmentally-friendly, and has low costs, high product safety and purity.