We present the development of a batch trapping, insulator-based dielectrophoretic (iDEP) device with three-dimensional design. The microfluidic devices use DC-biased, AC electric fields to selectively manipulate biological particles based on their electric properties. The mold for the polymer microdevices is fabricated using an RIE-lag technique which creates microchannels with varying depths using a single etch process. The resulting three-dimensional insulating constrictions permit operation at low applied voltages. By varying both the applied frequency and the ratio of AC to DC electric fields, the iDEP device can trap and separate polystyrene beads and E. coli cells.