Dielectrophoresis is a robust approach for manipulating bioparticles in microfluidic devices. In recent years, many groups have developed dielectrophoresis-based microfluidic systems for separation and concentration of various types of bioparticles, where the gradient of the electric field causes dielectrophoresis force acting on the suspended particles. Enhancing the gradient of the electric field with three-dimensional (3D) electrodes can significantly improve the efficiency of the system. Implementing planar electrodes in a 3D arrangement is a simple option to form a 3D-electrode configuration. This paper reports the development of a novel dielectrophoretic microfluidic system for continuously manipulating microparticles such as polystyrene microbeads and Saccharomyces cerevisiae cells. The fabrication process was relatively simple, cost-effective, and precise. Moreover, the device was tested to find the impact of various parameters on the concentration of polystyrene microbeads and separation of live and dead cells. The optimum working conditions, including flow rate, applied voltage amplitude, and frequency, were obtained accordingly. Furthermore, the experimentally observed trajectories of the particles agreed well with simulated counterparts. The device was able to efficiently and continuously perform with high throughput. Under an optimum condition, an efficiency of approximately 100% was obtained, confirming the capability of the proposed design with four triangular electrodes for continuous focusing and separation of live and dead cells as well as polystyrene particles.