The invention discloses an enzymatic refining process of chitosan, and belongs to the technical field of chitosan refining. The method comprises the following steps: adding keratinase into crude chitosan for enzymatic refining to obtain refined chitosan of which the protein content is lower than 0.2%, wherein the ratio of the use amount of the keratinase to the protein content in the chitosan is 1.1: 1-1.2: 1, the pH value of the enzymolysis reaction is 10-12 and the enzymolysis reaction time is 3-9 hours. The enzymolysis efficiency is improved, the content of protein in the crude chitosan isgreatly reduced, and the oxidation resistance of the chitosan is improved by optimizing the enzymolysis reaction temperature, the pH value, the enzyme adding amount and the reaction time. Protein in chitosan is removed by adopting specific keratinase so that the removal rate is high. Besides, the enzymatic reaction conditions are mild, the main chain structure of the chitosan is not influenced andchanged, the environment is not easily polluted and the process has very good practicability.