This paper presents a novel microfluidic dielectrophoresis (DEP) system to focus and separate cells of similar size based on their structural differences, which is more challenging than separation by size. Because, in this case, the DEP force is only proportional to the polarizabilities of cells. We used live and dead yeast cells as bioparticles to investigate the chip efficiency. Our designed chip consists of three sections. First, focusing cells in the center of the microchannel by employing a negative DEP phenomenon. After that, cells are separated due to the different deflection from high electric field areas. Finally, a novel outlet design was utilized to facilitate separation by increasing the gap between the two groups of cells. The proposed sheath-free design has one inlet for target cell injection requiring only one pump to control the flow rate, which reduces costs and complexity. Successful discrimination of the particles was achieved by using DEP force as a label-free and highly efficient technique. As an accessible and cost-effective method, soft lithography by 3D printed resin mold was used to fabricate microfluidic parts. Microchannel is made of Polydimethylsiloxane (PDMS) material that is biocompatible. The electrodes are made of gold due to its biocompatibility and non-oxidation, and a titanium layer is sputtered as the buffer layer for the adhesion of the sputtered gold layer to the glass. A standard microfabrication process is employed to create the electrode pattern. O2 plasma treatment yielded a leakage-free bonding between patterned glass and PDMS structure containing microfluidic channel. The maximum voltage applied to the electrodes (26 V) is lower than the threshold value for cell electroporation. Simulations and experimental results both confirm the effectiveness of the proposed microfluidic chip.