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This paper presents a high-throughput continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process. We design the continuous cell separation chip with three electrodes, where the cells in positive DEP affinity are separated from the central streamline. In the experimental study, we use the mixture of viable (live) and nonviable (dead) yeast cells as sample cells to be separated. We obtain the continuous cell separation conditions in the DEP affinity test: the sinusoidal electric fields of 5 MHz, 8Vp-p have been applied across the electrode array of 20 μm gaps immersed in the medium conductivity of 5 μS/cm. Using switched AC signal under these conditions, we continuously separate the yeast cells at the mixture flow rates of 0.1∼1 μl/min. The purity of the separated viable and nonviable yeast cells has been measured in the range of 95.9∼97.3% and 64.5∼74.3%, respectively.