With ribonucleic acid (RNA) as raw material, the enzyme catalytic hydrolysis reaction to generate the 5 '- adenosine (5 -' AMP), 5 '- guanosine monophosphate (5' - GMP), 5 '- cytidine acid (5' - CMP) and 5 '- uridine acid (5' - UMP party) and so on four kind of 5 'nucleotide, and then by chromatography separation to obtain high purity of four single 5' - nucleotide products. This product can be used in medicine, reagent, fine chemical industry, food additives and other fields.