A novel method for constructing an automated cell fractionation system was developed by integrating traveling-wave dielectrophoresis (twDEP) and laminar flow on a single biochip-based device. Novel electrodes designs on indium tin oxide (ITO) glass and pre-molded poly(dimethylsiloxane) (PDMS) microfluidic chamber, as well as an automated cell tracking method based on a computer-assisted image analysis method were designed and developed. The functioning of the device was demonstrated by isolating viable human myelogenous HL-60 cells from the dead ones. The simulation of the electric field and fluidic field, together with the experimental results confirmed our theoretical predictions. The preliminary data achieved with this device indicated that cells of different types could be effectively separated