Differentiation of normal human bladder cell (SVHUC) between two different-grade bladder cancer cell lines (TSGH8301, grade II and TCCSUP, grade IV) was successful developed based on a dielectrophoresis (DEP) microchip with microcavity array and multilayer electrodes. Single cell could be firstly trapped in the microcavity by negative DEP force between top and middle electrodes without overlapping problem; then, the trapped cells were sensed its impedance by sweeping AC signal in between middle and bottom electrodes. As the experimental results, the impedance of higher-grade bladder cancer cells was smaller than the value of lower-grade bladder cancer cells, i.e., TCCSUP (grade IV) < TSGH8301 (grade II), and the impedance of normal bladder cell was much higher than the values of both cancer cell lines. Basically, the impedances of all kinds of cell lines were decreased with the delay time measured when cells were taken out of the incubator. The largest difference of impedance between normal cells and cancer cells occurred as the delay time reached 1 hour, furthermore, the ratio of impedance between cancer cells and normal cells measured at 1 KHz and 0.2 V were 54% and 22% for TSGH8301 and TCCSUP, respectively. Consequently, the possibility of impedance measurement for evaluation of cancer cells was first proposed and investigated; moreover, the microchip provides the potential of electrical sensing for in vitro diagnosis under single cell resolution.