Gelatin, as natural macromolecular material, has been used in biomedical fields widely. In this study, various injectable gelatins A, B, and their compound AB microsphere-based gels (A-GMGs, B-GMGs and AB-GMGs) were prepared through water-in-oil emulsion method for hemostasis, and the effects of blood coagulation in vitro and surgical hemostasis (a deep liver wound model) in vivo were evaluated. Furthermore, the influences of gelatin sorts, the size of microsphere, zeta potential (ZP) and viscoelastic properties on hemostasis were also assessed. Results showed that the gelatin microspheres (GMs) exhibited smooth surface, good sphericity and the particle size of a rough normal distribution. GMs carried negative charges and their electronegativity was stronger than that of gelatin A (GA) and gelatin B (GB) raw materials. Rheological analysis showed that a decreasing particle size of the microspheres led to stronger gel strength, and solid-like gels were exhibited under low stress conditions and liquid-like gels were exhibited under high stress conditions. The blood clotting time of B-GMGs was within 60 s, which exhibited a significantly higher blood clotting effect compared with control groups. The hemostasis assay in vivo showed that the gels had better hemostatic effect on a deep liver wound bleeding model compared with control groups, especially B-GMGs. However, in vivo and vitro hemostatic experiments, particle size of GMs had no obvious influence on the hemostatic effect of the gels. In addition, the CCK-8 assay of bone marrow mesenchymal stem cells of murine (mMSCs) indicated non-cytotoxicity of GMs for cells. These results demonstrated that the gelatin microsphere-based gels (GMGs) had potential to be an effective hemostatic material for intracavitary and deep wound bleeding in surgery.