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Cell separation techniques are widely used in many biomedical and clinical applications for the development of screening, diagnosis and therapeutic tests. Current 3D microfluidics-based cell separation methods have limited applications in part due to low throughput and technical complexity. To address these critical needs, we have developed a 2D microfluidics surface which is the miniaturized version of a 3D microfluids cell separation device. Using low-frequency electric fields (1–10 Vpp and 1 kHz–20 MHz), we have first studied dielectrophoresis, AC electro-osmosis and capillary flow within a sessile drop, and finally utilized the results to develop the 2D cell separation surface. Our study has demonstrated that frequency-dependent dielectrophoretic force and AC electro-osmotic flow can be integrated to minimize the capillary flow and subsequently produce clusters of target cells within the 2D microfluidics surface. To demonstrate the concept, we have isolated the blood cells from a red blood cell-lysed blood sample. Cell isolation results show that significant improvement in throughout up to about 120-fold over 3D microfluidics devices. Additionally, due to the technical simplicity, this device offers great potential for use in a wide range of biomedical and clinical applications.