In this work, we present a device for cell manipulation and separation by using travelling wave dielectophoretic force. The device consists of a 16 parallel electrode array and microchamber. The dielectrophoretic PDMS chamber was fabricated by using standard microfabrication techniques. The Cr/Au parallel electrode array of 100 µm wide and 300 nm thick was patterned on a glass slide by sputtering through microshadow mask. The polystyrene microspheres suspension in de-ionized water and red blood cells in D-mannitol solution were used as tested cells. Cells respond to the electric field in various mechanisms depending on the applied voltage and frequency of the AC signals. For 4.5 µm polystyrene, the traveling wave dielectrophoresis happened when the applied voltage was 10 V, and the frequency of the applied signals was in the range of 50 kHz-700 kHz. For 10 µm polystyrene the twDEP occurred when the applied voltage was 7 V, and frequency was in the range 30 kHz-1MHz. While the red blood cells experienced the twDEP when the applied voltage was 3 V and frequency was in the range 50 kHz-2MHz. The mixed solution containing equal amount of 4.5 and 10 µm microspheres were used for separation test. The big microspheres were moved under twDEP force when the applied voltage was 7 V, and the frequency was in the range of 25 kHz-1MHz while the small microspheres were attached to the electrodes. Therefore, the twDEP device can manipulate and separate the microspheres with different sizes, and it can be further applied for cells selection.