Purpose: Hydroxyapatite (HA) is a promising scaffold material for bone defect treatment. This study aimed to evaluate the physicochemical and biological properties of a novel marine-derived HA extracted from blue fish scales and assess its potential as a bone substitute for clinical use. Methods: 80 female New Zealand white rabbits were assigned to 4 groups (n = 20): HA powder (EC group), 3Dprinted HA cylinder (I3DFFF group), negative control group (NC group; bone defect without biomaterial), and positive control group (PC group; bone defect treated with commercially synthesized HA). The biomaterials were characterized in vitro using X-ray diffraction, nitrogen adsorption, scanning electron microscopy, emission spectrophotometry, and MTT assays. In vivo evaluation involved the same techniques, supplemented with histological and radiological analyses at 1, 3, 6, and 9 months post-implantation. Molecular docking studies were also performed to investigate HA's effect on RANKL-induced osteoclastogenesis and the NF-kappa B signaling pathway. Results: In vitro characterization showed that marine-derived HA is a mesoporous material with a crystalline structure similar to natural bone. In vivo, the material demonstrated biocompatibility, osteoinductivity, and osteoconductivity, providing a supportive environment for bone regeneration. Molecular docking indicated that HA could inhibit osteoclastogenesis by suppressing the NF-kappa B signaling pathway. Conclusion: Our findings suggest that the two co-products, EC-17 and I3DFFF, possess the necessary physicochemical and biological characteristics to be developed as effective bone substitutes.