Cell characterization analysis usually involves a sequence of steps such as culture, separation, trapping, examination and recollection. In general, it is difficult to recover the identified cells and achieve a multi-run examination on a single chip for clinical samples. In the present study, a dielectrophoresis (DEP) micro-device was developed for multi-step manipulations of cells at the single-cell level. The structure of the DEP chip consisted of an indium tin oxide (ITO) top electrode, a flow chamber, a middle electrode on an SU-8 surface, a micro-cavity array of SU-8 and distributed electrodes at the bottom of the micro-cavities. The purpose of the three-layer-electrode design was threefold. First, cells could be trapped into the micro-cavities by negative DEP between the top and middle electrodes. After cells were trapped, cell analysis at the single-cell level could potentially be performed. This could include, for example, drug treatment or biomedical sensing on the chip without applying voltage. Once identified, the target cells could be individually released by controlling the bottom distributed electrodes. Finally, the rest of the trapped cells could be pulled out by a positive DEP force between the top and middle electrodes and flushed away for the next run of cell analysis. The multi-step manipulations of human bladder cancer cells (TSGH8301) were successfully demonstrated and discussed, providing an excellent platform technology for a lab-on-a-chip (LOC).