High-performance liquid chromatography (HPLC) is a powerful tool to separate and analyze complex samples. Monodiseperse porous silica microspheres (MPSMs) have been widely used as column packings in HPLC. However, synthesis of MPSMs with controllable sizes of both particles and pores for the separation of small solutes and proteins in HPLC still remains a challenge. In this paper, an effective and facile approach to prepare MPSMs with controllable particle size and pore size by using porous polymer microspheres as templates is presented. By employing porous PGMA/EDMA microspheres as templates and tetraethyl orthosilicate (TEOS) as the silica source, 5 μm MPSMs with tunable pore sizes were synthesized successfully. The as-prepared MPSMs were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), dynamic laser scattering, and mercury intrusion porosimetry. The results indicated that the MPSMs obtained retained the original size of the polymer templating particles and calcination caused almost no shrinkage. Furthermore, the effects of the pore size of polymer template microspheres, different amino-functionalizations of PGMA/EDMA microspheres and the mass ratio of PGMA/EDMA microspheres/TEOS on the pore size of MPSMs were carefully studied. The results indicated that the pore size of MPSMs was adjusted from 20 to 69 nm by controlling the pore size of the polymer microspheres and the mass ratio of PGMA/EDMA microspheres/TEOS in the sol-gel process. In addition, the amino-functionalization of PGMA/EDMA microspheres with different structure-directing agents, such as (3-aminopropyl)triethoxysilane (APTES), trimethylamine hydrochloride (TMA) and tetraethylenepentamine (TEPA), also resulted in MPSMs with the different pore sizes. MPSMs with large pore sizes of more than 30 nm were fabricated by using TEPA-functionalized PGMA/EDMA microspheres as templates, while with TMA-functionalized PGMA/EDMA microspheres as templates, MPSMs with pore sizes of approximately 10 nm were obtained. The as-prepared MPSMs were further modified with different silanes, such as C4, C8 and C18, to explore as stationary phases for the separation of proteins and small solutes in reversed phase liquidi chromatography (RPLC). The results illustrated that the baseline separation of 7 kinds of proteins could be achieved based on MPSMs with pore sizes of 30 nm, and 6 kinds of alkyl benzenes and 5 kinds of aromatic alcohol homologs could be separated completely based on MPSMs with pore sizes of 11 nm. This work demonstrated that MPSMs prepared by applying the polymer templating method showed a promising potential applicability in HPLC.