In this paper, an effective method of enriching polystyrene (PS) microspheres on dielectrophoretic chip by negative dielectrophoretic force was proposed. A star-shaped microelectrode was designed and fabricated on a glass substrate by a lift-off process. Under the action of a high-frequency AC signal, the star-shaped microelectrode chip can provide a specific non-uniform electric field, which can produce negative dielectrophoretic force.The effects of amplitude and frequency of AC signal, conductivity and concentration of working solution on the enrichment of PS microspheres were investigated. The results show that when the voltage parameter of AC signal is 9 V and the frequency parameter is 1 MHz, the PS microspheres with low conductivity are enriched in the center of the electrode. With the aid of negative dielectrophoresis force, the concentration of PS microsphere suspension in the range of 0.33~2.08 amol/mL can form microsphere clusters. The results of this study have potential applications in molecular cell biology, pharmacology, and biochemistry.