Integrating three-dimensional (3D) microelectrodes on microfluidic chips based on polydimethylsiloxane (PDMS) has been a challenge. This paper introduces a composite 3D electrode composed of Ag powder (particle size of 10 nm) and PDMS. Ethyl acetate is added as an auxiliary dispersant during the compounding process. A micromachining technique for processing 3D microelectrodes of any shape and size was developed to allow the electrodes to be firmly bonded to the PDMS chip. Through theoretical calculations, numerical simulations, and experimental verification, the role of the composite 3D microelectrodes in separating polystyrene particles of three different sizes via dielectrophoresis was systematically studied. This microfluidic device separated 20-, 10-, and 5-μm polystyrene particles nondestructively, efficiently, and accurately.