The present invention provides for a process for preparing a recombinant human thrombin. A process for preparing a recombinant human thrombin which comprises: (1) obtaining a transfectant cell producing a human prothrombin by introducing an expression vector, wherein a gene fragment coding for a human prothrombin gene is incorporated, into an animal cell; (2) purifying a human prothrombin from the culture of the transfectant cell above by an anion exchanger; (3) converting the purified human prothrombin into a human thrombin by subjecting said human prothrombin to the action of ecarin; and (4) purifying the human thrombin from the solution after treatment with ecarin by an affinity method using benzamidine and a cation exchanger, and human thrombin obtained by said process, and a CHO cell that produces human prothrombin.