Microfabrication technologies achieving precise manipulation of biological cells provide the potential for individual characterization, detection and assay to cells at the single-cell level. The main purpose of the present study was to develop a microfluidic chip with microwells for single-particle-based positioning by using electroosmotic flow. Therefore, the process could not only be reliable, but also simple without a syringe pump. A biocompatible material of polydimethylsiloxane (PDMS) was adopted as a structure in the microfluidic chip for single-particle-based array. The sample of 6 μL with latex particles (17 μm in diameter) was suspended in the sucrose medium with a concentration of 106 particles/mL and dropped into the microchannel for micropatterning. The DC (direct current) voltages for electroosmotic flow were set as 10, 15 and 20 volts, respectively. The velocity of electroosmotic flow increased with the applied voltages. The occupancy of particles decreased with voltages applied for both the microfluidic chips containing 20 or 30-μm microwells, which implied that the higher velocity of electroosmotic flow caused lower particulate occupancy. Furthermore, there was only one single particle within the individual microwell in most of occupied microwells with 20 μm in diameter, which was much higher than that for the 30-μm-diameter microwells. Micropatterned latex particles in microwells were successfully achieved in this preliminary study. The microfluidic chips with microwells with different diameters were fabricated herein, which was suitable for measurements at a single-cell level.