Provided are a microfluidic device for separating polarizable analytes via dielectrophoresis, the device including: a microchannel including a membrane having nano- to micro-sized pores; at lest two electrodes generating a spaciously non-uniform electric field in the nano- to micro-sized pores when an AC voltage is applied; and a power source applying the AC voltage to the electrodes, and a method of separating polarizable target materials using the device.