A two-step acid-base catalyzed sol-gel method has been used to prepare monodispersed poly(3-mercaptopropylsilsesquioxane) (PMPSQ) microspheres. Initially, the hydrolysis of 3-mercaptopropyltrimethoxysilane was catalyzed by hydrochloric acid in an aqueous medium, and then the addition of ammonium hydroxide initiated the condensation of hydrolysate. The formation process of PMPSQ microspheres was characterized by field emission scanning electron microscopy and Si-29 NMR. The formation mechanism of the monodispersed microspheres was also discussed. Through this method, monodispersed PMPSQ microspheres with a high yield were achieved and the particle size could be adjusted by changing the precursor concentration.