The present study reports a low-cost, new material, eco-friendly and reproducible microbes Bacillus subtilis mediated biosynthesis of TiO 2 nanoparticles. Titanium dioxide nanoparticles synthesized from titanium as a precursor, using the bacterium, B. subtilis. The synthesized nanoparticles were characterized and confirmed as TiO 2 nanoparticles by using the UV spectroscopy, XRD, FTIR, AFM and SEM analysis. The morphological characteristics were found to be spherical, oval in shape, individual nanoparticles as well as a few aggregates having the size of 66–77nm. The XRD shows the crystallographic plane of anatase of TiO 2 nanoparticles, indicating that nanoparticles structure dominantly correspond to anatase crystalline titanium dioxide.