Amphiphilic silica nanoparticles surface-functionalized by 3-aminopropyltriethoxysilane (APTES) and octyltriethoxylsilane (OTES) were successfully prepared and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectrometry (FT-IR) and thermogravimetry (TG) techniques. The potential use of these bifunctionalized nanoparticles as pseudostationary phases (PSPs) in capillary electrophoresis (CE) for the separation of charged and neutral compounds was evaluated in terms of their suitability. As expected, fast separation of representative aromatic acids was fulfilled with high separation efficiency, because they migrate in the same direction with the electroosmotic flow (EOF) under optimum experimental conditions. Using a buffer solution of 30mmol/L phosphate (pH 3.0) in the presence of 0.5mg/mL of the synthesized bifunctionalized nanoparticles, the investigated basic compounds were baseline-resolved with symmetrical peaks. Due to the existence of amino groups on the surface of nanoparticles, “silanol effect” that occurs between positively charged basic analytes and the silanols on the inner surface of capillary was greatly suppressed. Furthermore, the separation systems also exhibited reversed-phase (RP) behavior when neutral analytes were tested.