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Monodispersed mesoporous silica spheres (MMSSs) with tunable particle size were successfully synthesized from tetraethyl orthosilicate (TEOS) and cetyltrimethylammonium bromide (CTAB) under an alkaline condition. The MMSSs synthesized under different reaction conditions, such as reaction temperature, co-solvent amount, and TEOS and CTAB concentrations, have been investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), and nitrogen adsorption–desorption. The average particle size increases with increase in the reaction temperature from 40°C to 90°C, and 80°C is the optimal temperature to synthesize MMSSs with narrow particle size of 75 ± 14 nm. Ethanol plays a role as co-surfactant in low concentration, and then as co-surfactant and co-solvent in high concentration, which results in an increase in particle size from 75 ± 14 nm to 518 ± 30 nm and change of the mesoporous structure from paralleled array type to radially orientated type. The average particle size and yield of MMSSs increase with increase in the TEOS concentration when the TEOS concentration reaches saturated level (Cs = 0.09M), but some small silica particles appear when the TEOS concentration is higher than the critical level (Cs* = 0.134M). When the CTAB concentration increases, at the same TEOS concentration, the surface area, and pore volume increases, but the monodispersity becomes low.