The separation of the small colloidal particles from the mixture of two different sized particles using AC dielectrophoresis phenomenon was studied. The spherical mono-dispersed polystyrene particles dispersed in pure water were put into a perfusion chamber on a substrate, and AC electric fold was applied to the glass substrate with Au electrodes in 4 mm distance. The AC frequency was fixed at 1 kHz and the intensity of the field was varied from 25 V/cm to 160 V/cm. After applying the AC field, the degree of the chain formation that resulted from the particle movements by dielectrophoresis was observed by optical microscope. The mixture of the sized polystyrene particles at 0.5 vol% concentrations for each size was set in the dielectrophoresis conditions of 1 kHz and 100 V/cm. At this condition large sized polystyrene particles formed chains, on the contrary the sized polystyrene particles formed no chains. After water flowing for 20 min, it was found that small particles that were floating in the chamber had been removed by the water flowing.