Efficient separation and preconcentration of nanoparticles are crucial in a wide range of biomedical applications, particularly as target substances continue to diminish in size. In this study, we introduce an electric field-assisted membrane system that synergistically combines oversized-pore membranes with an electrokinetic particle retention mechanism. Utilizing Ti/Au-coated poly(tetrafluoroethylene) (PTFE) membranes, our approach generates electrokinetic forces to effectively separate and retain charged nanoparticles even smaller than the pores, achieving a separation efficiency over 99% and a preconcentration factor of 1.76 within 10 min. Additionally, membrane fouling and transmembrane pressure are significantly reduced compared to conventional filtration techniques, offering advantages such as lower driving pressure and improved particle recovery. Rigorous experimental analysis and theoretical modeling reveal that this method establishes a critical balance between drag and electrokinetic forces acting on the nanoparticles, thereby enhancing separation and concentration efficiencies. Our research outcome paves the way for advanced particle manipulation techniques, potentially transforming biomolecule enrichment practices in diverse biomedical fields, including point-of-care diagnostics, highly sensitive biochemical detection, and bioprocessing applications.