Polymethylsilsesquioxane (PMSQ) has become a kind of widely studied filler used in the electronic circuit board substrates due to its organic–inorganic hybrid structure, low dielectric constant, and good thermal stability, among other factors. Herein, the PMSQ microspheres were prepared by a two-step acid–base-catalyzed sol–gel method; the influences of reaction conditions including the ratio of water/methyltrimethoxysilane (MTMS), reaction temperature, concentration of the catalyst, and stirring time were systematically investigated; and the optimized reaction condition was then obtained towards a narrow particle size distribution and good sphericity. The microstructure of PMSQ microspheres was analyzed by the infrared spectrum and X-ray diffraction (XRD), which indicated that the as-prepared PMSQ had a ladder-dominant structure. The thermogravimetric analysis (TGA) demonstrated an excellent thermal stability of as-prepared PMSQ microspheres. More specifically, the dielectric constants at high frequency (1~20 GHz) of as-prepared PMSQ microspheres were measured to be about 3.7, which turned out a lower dielectric constant compared to SiO2 powder (≈4.0). This study paves the way to further improve the performance of the electronic circuit board substrates for the application of high-frequency electronic packaging.