Abstract: The present disclosure provides a preparation method of a 2?-substituted pyrimidine nucleoside, including the following steps: subjecting a compound of Formula I or Formula II to a series of process including dehydration, selective 5?-protection, ring-opening reaction using magnesium alkoxide and deprotection to obtain a 2?-substituted pyrimidine nucleoside of Formula VII or Formula VIII. In the present disclosure, the preparation method has desirable universality for different substrates; the ring-opening with a protected anhydrous pyrimidine nucleoside improves a solubility of the substrate, with milder reaction conditions than those of a traditional synthetic route; the formation of a dimer is avoided during the ring-opening to improve the yield; in addition, an intermediate (5?-O-bis-p-methoxytrityl-protected 2?-substituted pyrimidine nucleoside) can be directly used in synthesis of a corresponding phosphoramidite monomer, with a wider potential for use.