The polymerization-induced colloid aggregation (PICA) method is commonly used to create SiO2@SiO2 core–shell silica microspheres (CSSMs), but it often encounters challenges such as incomplete shell coating and poor reproducibility. In this paper, the formation mechanism of the silica shell layer during the preparation of SiO2@SiO2 CSSMs using the PICA method was investigated. It was found that ureido modification can reduce the Zeta potential of the silica core surface, facilitating the deposition of coacervates formed by urea-formaldehyde resin (UF) and silica nanoparticles on the silica core surface to form the SiO2 shell layer when the Zeta potential of the surface is in the range of −20.1 mV to −4.8 mV. By controlling the interfacial state and surface potential of the SiO2 core, the issue of inconsistent reproducibility in preparing SiO2@SiO2 CSSMs can be overcome. Furthermore, optimization of experimental parameters such as pH, reaction temperature, water content, and reaction time can enhance the formation process. The thickness of the shell and pore size of CSSMs can be successfully adjusted by varying the reaction time and the particle size of colloidal silica sol, respectively. With optimal conditions, the semi-scale production yield of SiO2@SiO2 CSSMs can be increased to 50 g. The SiO2@SiO2 CSSMs were modified with octadecyltrichlorosilane (ODS) to be used as stationary phases in reversed phase liquid chromatography (RPLC) for fast separation of small solutes, peptides, and proteins. The superior separation efficiency indicates that the improved PICA method has the potential to be utilized as an alternative to the layer-by-layer method for large-scale production of SiO2@SiO2 CSSMs.