To improve chitosan hemostasis, carboxylated sisal fiber and kaolin were introduced to obtain carboxylated sisal fiber/chitosan/kaolin (SF/CS/K) composite sponges (the weight ratio of 3: 3:4, 4:4:2, 5:5:0) by freeze-drying method. The results showed that the ionic cross-linking of the carboxylated sisal fiber with chitosan and kaolin-loading endowed the composite sponges with not only oriented groove porous structure, high mechanical strength, porosity, water absorption and compress recovery, but also suitable biodegradation, good cytocompatibility, hemocompatibility, protein adsorption, antibacterial activity. Especially, compared with commercial gelatin hemostatic sponges, the composite sponge of SF/CS/K displayed better coagulation ability and hemostatic effect, and animal experiments further demonstrated that the bleeding amount and hemostatic time of SF/CS/K were greatly reduced in rat hemostatic models of tail amputation, femoral vein trauma and liver injury, owing to the synergistic hemostatic effect of chitosan, kaolin and sisal fiber, as well as the groove porous structure, which endowed them with strong adhesion for red blood cells. Conclusively, SF/CS/K composite sponge had the best hemostatic effect because of the most appropriate component ratio, which is a novel promising natural hemostatic sponge for effective and rapid hemostasis in deep massive bleeding sites.