Biomaterials are playing a pivotal role in facilitating hemostasis and regeneration. Fabrication of cuttlefish bone derived hydroxyapatite (HAp) infused alginate composite membrane for enhanced hemostasis and tissue regeneration. Cuttlefish bone HAp revealed Ca5(PO4)3(OH) and Ca3(PO4)2 crystalline phases with elongated spherical morphology. This HAp infused alginate membrane was fabricated using lyophilization, carboxylic as well as phosphate groups (HAp) was confirmed the formation of membrane. Graded as well as porous morphology was observed in pure alginate (ALG) and HAp alginate respectively. Enhanced hydrophilic (28.44°) and mechanical properties (23.7 MPa) were observed in HAp-ALG membrane. Optimal compatibility features was observed with the treatment of blood and fibroblast cells. Cuttlefish bone derived HAp-ALG membrane revealed rapid hemostatic behavior that was observed by clot initiation and morphological analysis. Physico-chemical, mechanical, and biological properties of cuttlefish bone derived HAp-ALG are ascertain its suitability for regeneration and hemostatic applications.