Over the last decades, hydroxyapatite (HA) and its composite with biopolymer have been extensively developed and applied in biomedical application. The aim of this study is to produce a novel 3D porous HA scaffolds for bone tissue regeneration. Scaffolds with varying composition of HA, chitosan, gelatin, agarose and poly vinyl alcohol (PVA) were prepared using freeze drying method. The composite scaffold was analyzed to determine density, porosity, biodegradability, swelling kinetics, morphology and structural properties. Porosity and density of the prepared scaffolds were 85.17 to 92.21% and 0.317 to 0.495 g/cm3, respectively. The swelling ability of the prepared scaffolds showed similar efficacy. FTIR analysis showed intermolecular interaction between components in the scaffold. Pore size of the developed scaffolds were measured by scanning electron microscopy and found that regardless of their composition showed adequate pore sizes ranging from 174 to 405 µm. Brine shrimp lethality assay indicated that the obtained scaffolds had no cytotoxic effects, and that they had good biocompatibility. The results suggested that, these homogeneous composite scaffolds were found to be potential candidates as bone grafting materials for bone tissue engineering applications.