In this work, multilayer zirconia-coated silica (ZrO2/SiO2-n) microspheres were successfully produced by a straightforward hydrothermal procedure with a low concentration of Zr4+ (5 mM) under low potential of hydrogen (pH) conditions (pH = =2). The obtained ZrO2/SiO2-n materials exhibited favorable characteristics for high-performance liquid chromatography (HPLC) separation, including high surface area and pore volume, good pore structure, narrow particle size, and pore size distribution. In addition, the zirconia coverage in the mesopores was confirmed by soaking the material in 1 M NaOH solution, with the particles showing strong resistance to the basic solution. The obtained ZrO2/SiO2-n stationary phases were packed into a fused-silica capillary tubing for the separation of alkaloids in hydrophilic interaction chromatography (HILIC) mode, and a column efficiency of 47,800 plates/m was obtained for berberine on a ZrO2/SiO2-6 micro column. The ZrO2/SiO2-6 microspheres were further modified by dodecylphosphonic acid (C12P-2-ZrO2/SiO2-6); the C12P-2-ZrO2/SiO2-6 material showed great potential for application in reversed-phase liquid chromatography (RPLC) mode. The C12P-2-ZrO2/SiO2-6 micro column showed a column efficiency of 55,000 plates/m for naphthalene and 51,300 plates/m for benzene.