This paper reports a facile method for fabricating monodispersed mesoporous bioactive glass sub-micron spheres (MBGS) using dodecylamine (DDA) as a catalyst and template agent in sol–gel process. The effects of synthesis conditions including the amount of DDA, temperature of hydrolysis and the volume ratio of alcohol to water (AW ratio) on the resulting particle size, morphology, monodispersity and pore size distribution of MBGS are investigated and discussed. The results indicate that the particle size, morphology, monodispersity and pore size distribution of MBGS depend on the amount of DDA, the temperature of hydrolysis and the AW ratio. Meanwhile, using DDA as the structure directing agent and hydrolysis catalyst under optimal synthesis conditions (e.g. 4 g DDA, hydrolysis temperature at 40 °C and AW ratio at 4) is in favor of obtaining MBGS with mesoporous surface structure, large specific surface area (362.073 m2 g−1), relatively homogeneous particle size (~560 nm) as well as good apatite-forming activity. The unique structure and properties may turn MBGS into a good candidate as a drug delivery carrier or an injectable biomaterial for bone tissue regeneration.