Monodisperse SiO2 microspheres have found applications in catalysis, drug delivery, coatings, cosmetics, optical sensing and plastics. The particle size of monodisperse SiO2 microspheres is closely related to its application. In this paper, monodisperse SiO2 microspheres with tunable diameter were successfully synthesized using cetyltrimethylammonium bromide (CTAB) as template. The monodisperse SiO2 microspheres with diameters ranging from 200 nm to 3 μm were obtained by controlling the concentration of CTAB, tetraethyl orthosilicate (TEOS), diethanolamine (DEA) and reaction temperature. The BET surface area could reach 835 m2•g-1 and mean pore diameter was 2.3 nm. The formation mechanism of monodisperse SiO2 microspheres was investigated.