The present study aimed to preparation of hydroxyapatite using strontium (Sr2+) and chitosan (CS) for the preparation of porous scaffold material like SrHA-CS. These synthesized nano-materials were alsofurther characterized by Fourier transform infrared spectroscopy and X-ray diffraction analysis to confirm the phase purity and crystallinity. The crystal-like morphology of the SrHA-CS was observed using a high-resolution transmission electron microscope (HRTEM) which revealed rod-like morphology with the major elements confirmed by energy-dispersive X-ray spectroscopy (EDX). The size of the synthesized nanomaterial and their stability were analyzed through dynamic light scattering (DLS)and zeta potential. The hemolytic study indicates that SrHA-CS is non-toxic red blood cells of the humans. Besides, SrHA-CS is also active on disease-causing microorganisms and exhibit a strong zone of inhibition. The cytotoxic (MTT) assay systemically investigatedthe synthesized samplesagainst SaOS-2 human osteosarcoma cell lines it resulting in effectively induce cell proliferation without generating any harmful effect. The acridine orange/Ethidium bromide dual staining (AO/EB) under the fluorescence microscope data suggests that apoptotic cell nuclei became irregularly changed. Our present study findings concluded that the strongly recommend the developed SrHA-CSscaffold excellent materialhas mechanical and functional properties including biological activities while compared to other synthetic nano-materials. Hence, the synthesizedSrHA-CSmaterial can be considered as a promising cost-effective nano-material for human bone regenerative applications.