Extracellular vesicles (EVs) play a crucial role in diagnosis and treatment, yet obtaining highly purified EVs from complex biological samples is often hindered by nanoscale contaminants. In this work, considering the charge-to-size characteristics of EVs, a circular multicavity electrophoresis (CME) with gradient pore size distribution was constructed in the gradient electric field to realize the isolation and preparation of EVs. By the gradient gel sieving effect, small cell debris, EVs, and proteins in biological samples were gradually separated. The integration of ultrafiltration (UF) with CME synergistically enhances EV purification and preparation, resulting in a purity level 3.15 times higher than that achieved through ultracentrifugation (UC). The high yield preparation of EV was achieved through continuous injection facilitated by the application of a gradient electric field, where 3.55 × 10 ± 6.32 × 10 particle numbers mL of EVs were prepared from 36 mL of cell supernatant, and the recovery approached 87.65 ± 9.03%. Further evaluation of the cell uptake efficiency of EVs derived from umbilical cord mesenchymal stem cells prepared by CME-UF revealed that this approach effectively preserves both the integrity and bioactivity of the EVs. This work presents a novel approach for the isolation and preparation of EVs, offering valuable insights into future biological studies.