The invention discloses a purification process for thrombin. The purification process comprises a step of nano filtration; before nano filtration is performed, a membrane bag is washed and filtered to remove impurity protein of which the molecular weight is greater than 200KD. According to the purification process disclosed by the invention, macromolecular proteins in a thrombin solution can be removed by washing and filtering the membrane bag, so that the condition that a nano-membrane pore channel is blocked by macromolecular substances is avoided; in addition, filtering conditions of the membrane bag are mild, so the condition that the activity of the thrombin is reduced is avoided, and the aim of removing non-lipid-enveloped viruses is achieved.