Outer membrane vesicles (OMV) are bioactive extracellular vesicles(EVs,but their low yield and purity limit their widespread application. This study found that adding the sterile supernatant of Escherichia coli to a mixed solution of Polyethylene Glycol (PEG) and Ammonium Sulfate ((NH₄)₂SO₄) rapidly generated a precipitate. Upon standing, the PEG / (NH₄)₂SO₄ system formed an aqueous two-phase system(ATPS）, with the precipitate enriched at the phase interface. Its efficiency for OMV separation was significantly higher than that of ultracentrifugation (UC). Based on this discovery, we designed an OMV purification process: ultrafiltration concentration - separation (UC or aqueous two-phase precipitation (ATPP)) - size-exclusion chromatography (SEC). This process maintained high recovery while removing impurities and residual precipitants generated by ATPP. OMV obtained through this process using ATPP method showed a 5-fold higher yield compared to those enriched by UC, while also exhibiting intact morphology and uniform distribution. Furthermore, we applied this method to extract OMV from Neisseria meningitidis serogroup B, a strain with application in OMV vaccines, and successfully obtained the corresponding OMV, indicating the promising application potential of this method.