Cobalt ferrite (CoFe2O4) has numerous desirable features such as high anisotropy, good magnetic saturation, large coercivity and high mechanical strength. These remarkable characteristics provide extensive employment for their wide application from industrial to biomedical. The present study aims to evaluate the crystal properties, particle size, and morphology of CoFe2O4 nanoparticles prepared by the thermal treatment method and investigates their biocompatibility. XRD and FTIR analyses demonstrated that CoFe2O4 nanoparticles have an inverse cubic spinel crystalline structure. The average size of spherical CoFe2O4 nanoparticles was confirmed by SEM analysis as 58 nm. The cytotoxic effect of CoFe2O4 nanoparticles was evaluated in L929 cells by using the XTT cell viability assay. After 24 h treatment of CoFe2O4 nanoparticles with a concentration between 10 and 1000 mu g/ml, the viability of L929 cells did not significantly decrease. It is concluded that growth CoFe2O4 nanoparticles are beneficial materials for use in various biomedical applications.