Bone injuries and defects are therapeutically challenging. Autografts and allografts are ideal treatment approaches for bone regeneration, but, they are coupled with a number of complications, and variable outcomes. Bone substitute biomaterials, have shown great potential for bone tissue engineering application. Which not only efficiently solved the problem of limited supply, but also provided possibility for reducing immune-complications and enhanced bone formation. Hydroxyapatite (HA) has been used in clinical bone graft procedures for more than 25 years. However, its poor tensile strength and fracture toughness compared with bone make it unsuitable for major loadbearing devices. Carbon nanotubes (CNTs), with their high aspect ratio and excellent mechanical properties, have the potential to strengthen and toughen Hydroxyapatite (HA) without offsetting its bioactivity, thus it has been a significant challenge to the application of HA for the replacement of load-bearing and/or large bone defects. The main aim of the study is to functionalized the MWCNT to attach functional groups to the nanostructure. These functionalized nanoparticles then reinforce with HA to make MWCNT-HA nanocomposites. Further, these nanocomposites are tested for its biocompatibilities with mouse fibroblast cell lines L929 in vitro. The functionalization of MWCNTs with acid mixture appends the functional groups to the structure, called as functionalized MWCNTs. These functionalized CNTs is now ready to interact with other materials to form a composite. We reinforced these functionalized CNTs with HA in situ, that leads to synthesis of MWCNT-HA composite. Furthermore, this study investigates the cytotoxic effects of functionalized Multiwalled Carbon nanotubes (f-MWCNTs) and its composites with HA in L929 mouse fibroblast cell line. FTIR analysis confirms the attachments of different functional group e.g., –C–O–, C O, –COOH to the composite. The FESEM image confirms the physical interaction of HA and CNT. The Interaction of these material with L929 cell lines shows no detrimental effects.