Poly(butylene succinate) (PBS) and gelatin-coated PBS electrospun membranes are evaluated for use as support materials to immobilize thrombin, an effective hemostat for topical injury, and three methods differing in whether and when gelatin is included are envisaged to prepare thrombin-loaded PBS-based electrospun membranes for use as rapid hemostatic materials. Both PBS and gelatin-coated PBS membranes have high porosity, excellent wettability, rapid water penetration rate, and high water uptake, and thus are suitable support materials for thrombin. The thrombin immobilized onto gelatin-coated PBS membrane has both high initial enzyme activity and high storage stability ascribed to the stabilizing effect of gelatin on thrombin activity. The hemostasis performance of thrombin-immobilized membrane is evaluated in a rat liver model, showing shorter hemostasis time and less blood loss than clinically used gelatin sponge. The hemostasis mechanism is attributed to combined effects of thrombin and porous structure of electrospun membrane.