The invention discloses a preparation method of 3-hydroxymethyl cefotaxime, which is characterized by comprising the following steps: adding cefotaxime to purified water at room temperature, adjusting the pH, adding cephalosporin C deacetylase, and maintaining the pH when the pH is maintained. When unchanged, the cephalosporin C deacetylase is filtered off, the filtrate is collected, the mixed solution of an inorganic acid and an organic solvent is added dropwise, the pH is adjusted to crystallize, the crystal is grown, filtered, and dried to obtain 3-hydroxymethylcefotaxime. The invention uses cefotaxime as raw material, adopts biological enzyme method, avoids the use of chemical synthesis methods, has few types of solvents, low toxicity and environmental protection, mild reaction conditions, simple steps, high product conversion rate, and simple and easy-to-control operations. The yield of the target product can reach 74%, and the purity can reach up to 97.6%. It fills the blank of preparing the impurity by biological enzymatic synthesis method, which is beneficial to the subsequent structure analysis and pharmacological research, and has important theoretical significance and practical application value for improving the quality of cefotaxime sodium and reducing the risk of clinical drug use.