The nanocomposite of nano-hydroxyapatite/bacterial cellulose (nHA/BC) obtained by depositing in simulated body fluid (SBF), incorporating their excellent mechanical and biological properties, is expected to have potential applications in bone tissue engineering. However, the biological response evaluation of biomaterials is required to provide useful information to improve their design and application. In this article, the in vitro cytotoxicity of composites nHA/BC as well as its degradation residues was studied. Scanning electron microscopy (SEM) was used to observe the morphology of original materials and their degradation residues. The degree of degradation was evalued by measuring the concentration of reducing sugar (glucose) by ultraviolet spectrophotometer. Bone-forming osteoblasts (OB) and infinite culture cell line L929 fibroblasts were used to measure the cytotocixity of materials with MTT assay. Both kinds of cells in infusion proliferate greatly in a normal form and their relative growth rate (RGR) exceeds by 75%, which shows the cytotoxicity of materials is graded as 0~1, according to the national standard. Nevertheless, bone-forming OB cells, as a kind of target cells, are more susceptive on the cytotoxicity than infinite culture fibroblast cells L929. The results suggest the nanocomposite of nHA/BC without cytotoxicity is greatly promising as a kind of scaffold materials for bone tissue engineering and tissue functional cells are more suited to evaluate the cytotoxicity of biomedical materials.