This study proposes a microfluidic device capable of separating monocytes from a type of cancer cell that is called T-cell acute lymphoblastic leukemia (RPMI-8402) in a continuous flow using negative and positive dielectrophoretic forces. The use of both the hydrodynamic and dielectrophoretic forces allows the separation of RPMI-8402 from monocytes based on differences in their intrinsic electrical properties and sizes. The specific crossover frequencies of monocytes and RPMI-8402 cells have been obtained experimentally. The optimum ranges of electrode pitch-to-channel height ratio at the cross sections with different electrode widths have been generally calculated by numerical simulations of the gradients of the electric field intensities and calculation their effective values (root-mean-square). In the device, the cell sorting has been conducted empirically, and then, the separation performance has been evaluated by analyzing the images before and after dielectrophoretic forces applied to the cells. In this work, the design of a chip with 77μm gold-titanium electrode pitch was investigated to achieve high purity of monocytes of 95.2%. The proposed device can be used with relatively low applied voltages, as low as 16.5V (peak to peak). Thus, the design can be used in biomedical diagnosis and chemical analysis applications as a lab-on-chip platform. Also, it can be used for the separation of biological cells such as bacteria, RNA, DNA, and blood cells.