The aim of the study was to explore the polyethylene glycol–dextran two‐-phase polymer system formed in human plasma to isolate the exosome‐-enriched fraction of plasma extracellular nanovesicles (ENVs). Systematic analysis was performed to determine the optimal combination of the polymer mixture parameters (molecular mass and concentration) that resulted in phase separa-tion. The separated phases were analyzed by nanoparticle tracking analysis and Raman spectros-copy. The isolated vesicles were characterized by atomic force microscopy and dot blotting. In con-clusion, the protein and microRNA contents of the isolated ENVs were assayed by flow cytometry and by reverse transcription followed by quantitative polymerase chain reaction (RT‐-qPCR), re-spectively. The presented results revealed the applicability of a new method for plasma ENV isolation and further analysis with a diagnostic purpose.