The need to develop bioactive ink for 3D printed biomedical products have posed challenges to both researchers and scientists. This study aimed to synthesize HAp ink from Nile tilapia (Oreochromis niloticus) fish scales for 3D-printed bone scaffolds and to investigate the bone sample’s mechanical, structural, and microstructural properties. Moreover, an economically viable and environmentally friendly method, freeze drying method was utilized for the manufacturing of hydroxyapatite. The X-ray diffraction (XRD) results revealed that the Hap powder synthesized had more excellent crystal dimensions than the standard HAp, and the material had a high level of crystallization. The scanning electron microscopy images exhibited a dense aggregation of closely spaced and uniformly distributed micropores. The mechanical tests showed that the structure retains its strength even after reaching a maximum stress of 16.32 MPa, demonstrating a satisfactory degree of toughness for a scaffold. FTIR investigations confirmed the presence of functional groups associated with the synthesis of hydroxyapatite (HAp). When synthetic materials were immersed in simulated body fluid (SBF), their FTIR spectra showed a decrease in the intensity of the distinctive peaks related to carbonate (B-type). The study revealed that the scaffold had a uniform arrangement of small holes, facilitating improved penetration of SBF fluid. Within biomedical applications, this signified that the human body would adapt to the implanted biomaterial faster.