A method for a synthesis of anhydrothrombin is provided which features a short process, an easy procedure, and high yields.The method comprises(A) a step of causing an active serine residue site of thrombin to react with an inhibitor,(B) a step of performing an alkali treatment at a pH of not less than 11, and(C) a step of performing an operation of recovery, and carries out these steps sequentially in the order mentioned, and is characterized by causing at least the step of performing the operation of recovery to proceed in the presence of at least one compound selected from the group consisting of polyhydric alcohols and saccharides, and a salt or an amphoteric electrolyte.