The invention discloses a preparation method of 16alpha-hydroxyprednisolone, which comprises the following steps: by using a compound I prednisone acetate as an initial raw material, carrying out esterification reaction, reduction reaction, elimination reaction, oxidation reaction and hydrolysis reaction to obtain the end product VI 11beta,16alpha,17alpha,21-tetrahydroxyprogsteroid-1,4-diene-3,20-dione, i.e. 16alpha-hydroxyprednisolone. By using the cheaper initial raw material, the method has the advantages of shorter reaction steps, feasible reaction steps, higher yield and higher economy and safety for production, and is more suitable for industrial production. The method avoids using the traditional virulent product for bis-hydroxy oxidation, enhances the reaction safety and operability, simplifies the multiple protection and deprotection operation steps, greatly shortens the synthesis route, lowers the production cost, basically prevents the three-site carbonyl groups from being simultaneously reduced in the reduction process, greatly reduces the side reactions, and enhances the yield and quality of the reduction reaction.