Micropatterning has been applied in pharmaceutical research and drug discovery as an effective tool. Assays and tests can be easily performed by arranging microparticles in an array. However, the quality of the microparticle pattern influences the reliability of the results. In this study, an automatic single-microparticle patterning system was developed. This system enables precise patterning of single microparticles through dielectrophoresis, which can manipulate micro-objects (e.g., bead, protein, and cell). Orange fluorescent polystyrene beads (40 μ m) were suspended in 6-aminohexanoic acid solution. In contrast to the conventional microfluidic configuration, electrode-based microchip suspended above the substrate can selectively trap and pattern the microbeads. In particular, the microbeads laying on the substrate can be displaced to different positions relative to the patterning electrodes in the microchip. A vision-based approach was used to evaluate necessary information such as the gap distance and positions of the electrodes and microbeads in the image. Experiments were performed to examine the strategy used to construct high-quality single-bead patterns. With the proposed system, different single microbead patterns can be successfully constructed on a glass substrate. Results confirmed that this system offers an automatic method with high flexibility to construct different single microparticle patterns for various applications.