A series of anionic surfactant templated mesoporous silica (AMS) monodispersed nanospheres were synthesized by anionic surfactant, co-structure-directing agent (CSDA) bearing amino group, and silica source in the presence of nonionic surfactant. The amino group functionalized well-ordered cubic Fd-3m, bi-continuous cubic Ia-3d and disordered structures with tunable pore size from 2.2 to 9.2nm and the particle size from 30 to 320nm, have been synthesized by changing the type of the surfactant, the concentration of the nonionic and anionic surfactant and the reaction temperature. Loadings of the amino groups were determined in the range of 2.7–3.4mmol/g. It has been found that, the higher concentration of the nonionic and the anionic surfactant, the shorter chain length of the anionic surfactant and the higher reaction temperature favor the formation of mesoporous silica nanospheres with larger particle size. These functional AMS nanospheres may find potential use in catalysis, sensor devices and bio-applications, etc.