Up to now, the chromatographic separation and analysis of highly polar compounds are still challenging because of low retention ability and selectivity of conventional stationary phases (SPs). Developing new SPs to achieve high-resolution and high-efficiency separation of polar compounds is of great importance for health and medicine, food safety, environmental protection, etc.. Herein, an influential multi-step modification strategy (modification with 1H,1H,2H,2H-perfluorooctyltriethoxysilane on the silica twice and end-capped with trimethylchlorosilane once) is introduced to prepare C8F13-SiO2 SP. The physical properties of C8F13-SiO2 SP, including morphologies, pore structures and carbon content were fully characterized by various instrumental techniques. The packed HPLC column with C8F13-SiO2 SP exhibited high column efficiency, good peak shape (tailing factors for most analytes are between 0.9 and 1.3), and resolution for simultaneous separation of ginsenosides, sulfonamides, nucleosides, and benzoylurea insecticides (BUs); meanwhile, the retention mechanism of typical reversed-phase liquid chromatography (RPLC) mode was observed. A column efficiency of 30,220 plates/m for separation of ginsenoside Re was obtained. Furthermore, the resulting columns benefit from the advantages of supreme reproducibility, repeatability and stability. Importantly, the chromatographic properties (e.g. column efficiencies, chromatographic peak shapes, retention ability and resolution) of the C8F13-SiO2 SP for the polar analytes were superior to those of the commercial SPs (including commercial amino-modified silica SP, diol-modified silica SP and commercial PFP-SiO2 SP). Thus, the C8F13-SiO2 SP is able to satisfy the strict requirements of highly polar compounds separation.