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Bone scaffolds mimicking the three-dimensional bone structure are of essential importance for bone regeneration. The aim of this study was to develop and optimize the production method of highly macroporous bone scaffold composed of polysaccharide matrix (chitosan–agarose) reinforced with nanohydroxyapatite. The highly macroporous structure was obtained by the simultaneous application of a gas-foaming agent and freeze-drying technique. Fabricated variants of biomaterials (produced using different gas-foaming agent and solvent concentrations) were subjected to porosity evaluation and compression test in order to select the scaffold with the best properties. Then, bioactivity, cytotoxicity, and cell growth on the surface of the selected biomaterial were assessed. The obtained results showed that the simultaneous application of gas-foaming and freeze-drying methods allows for the production of biomaterials characterized by high total and open porosity. It was proved that the best porosity is obtained when solvent (CH3COOH) and foaming agent (NaHCO3) are applied at ratio 1:1. Nevertheless, the high porosity of novel biomaterial decreases its mechanical strength as determined by compression test. Importantly, novel scaffold is non-toxic to osteoblasts and favors cell attachment and growth on its surface. All mentioned properties make the novel biomaterial a promising candidate to be used in regenerative medicine in non-load bearing implantation sites.