The invention relates to chitosan-based styptic sponge with a thrombin immobilization effect, and the chitosan-based styptic sponge is a porous sponge made from chitosan with the thrombin immobilization effect and hemostatic. The preparation method of the chitosan-based styptic sponge provided by the invention comprises the following steps: immobilizing thrombin with chitosan or carboxymethyl chitosan, and adding other styptics, cryoprotectants and crosslinking agents to prepare the porous styptic sponge, wherein the weight ratio of the chitosan or carboxymethyl chitosan to the thrombin is 100:(0.1-20); pre-freezing, lyophilizing in vacuum, casting, cutting, encapsulating and sterilizing to prepare the chitosan-based styptic sponge. According to the chitosan-based styptic sponge, the thrombin is immobilized through the chitosan or carboxymethyl chitosan, so that the stability and procoagulant activity of the thrombin can be improved, and the property of the prepared chitosan-based styptic sponge is more stable, the procoagulant and wound healing effects are remarkably improved, and the chitosan-based styptic sponge can be widely applied to wound or surgery hemostasis.