The invention discloses a preparation method of 5'-nucleotide, comprising the following steps of: suspending nucleoside in an organic solvent containing tricresyl phosphate low-level alkyl ester; adding a catalyst; and then contacting an obtained suspension with a phosphorylation reagent at the temperature of -30-20 DEG C so that the nucleoside is phosphorylated to obtain the 5'-nucleotide, wherein the weight ratio of the nucleotide and the catalyst is 100:0.5-1.5. The method can be used for preparing the high-purity high-yield 5'-nucleotide, shortens the time of the phosphonation reaction ofthe nucleotide, is easier to eliminate the impurities during the purification of the nucleoside and reduces the difficulty of post process.