Hemostasis is a major challenge in surgical procedures and traumas. Conventional hemostatic methods have limited efficacy and may cause additional tissue damage. In this study, we designed a novel hemostatic agent based on the in situ gel formation of gelatin cross-linked by a novel microbial transglutaminase(mTGase), in which the amino acid sequences differed from commercial mTGases. The new hemostatic agent showed the same biochemical crosslinking chemistry as the final stages of the blood coagulation cascade while using gelatin as a “structural” protein(rather than fibrin) and a calcium-independent mTGase as the crosslinking catalyst(rather than factor XIIIa). In rat liver hemostasis models, the hemostatic agent not only showed a similar hemostatic effect as that of SURGIFLO~(positive control), but also stronger adhesion strength and elasticity than SURGIFLO~.Therefore, this biomimetic gelatin-mTGase mix hemostatic is a novel and effective surgical sealant. Hemostasis is a major challenge in surgical procedures and traumas. Conventional hemostatic methods have limited efficacy and may cause additional tissue damage. In this study, we designed a novel hemostatic agent based on the in situ gel formation of gelatin cross-linked by a novel microbial The new hemostatic agent showed the same biochemical crosslinking chemistry as the final stages of the blood coagulation cascade while using gelatin as a “structural ” protein (rather than fibrin ) and a calcium-independent mTGase as the crosslinking catalyst (rather than factor XIIIa). In rat liver hemostasis models, the hemostatic agent not only showed a similar hemostatic effect as that of SURGIFLO ~ positive control, but also stronger adhesion strength and elasticity than SURGIFLO ~ .Therefore, this biomimetic gelatin-mTGase mix hemostatic is a novel and effective surgical sealant.