<p>Single-cell analysis is crucial for understanding the specificity of individual cells, yet its advancement is limited by the technical challenges of precise single-cell manipulation. Microfluidics has made significant advancements in single-cell manipulation, making it a powerful tool for analysis. This study presents a dielectrophoresis-assisted microfluidic device for single-cell manipulation. By coupling flow and electric fields, this device enables single-cell focusing, along with fixed-frequency capture and release on a microfluidic chip. This study employs theoretical and finite element method analysis to determine the cell dielectric parameters (K562), medium dielectric parameters (<i>σ</i>_m = 55 mS/m, <i>ε</i>_m = 7.08 × 10⁻¹⁰), flow field parameters, and electric field parameters. Cell focusing and periodic cell capture and release were successfully achieved in FEM analysis. Theoretical parameters were further optimized experimentally, resulting in a single-cell capture efficiency exceeding 98%. By coordinating the flow and electric fields, the system successfully achieved controlled single-cell capture and release at a fixed frequency. This work provides a flexible approach for precise single-cell manipulation in microfluidic chips. This device has significant potential for applications in single-cell analysis, cell biology research, early disease diagnosis, personalized medicine, and droplet microfluidic single-cell encapsulation.</p> Graphical Abstract <p></p>