Background. MicroRNAs (miRNAs) circulating in plasma are promising markers for the diagnosis of malignant tumors, including prostate cancer. However, the existing techniques used for their detection fail to ensure sufficient diagnostic accuracy. One of the possible ways to improve it is to isolate membrane nano-sized extracellular vesicles (nsEVs) secreted by prostate cells. Presumably, the analysis of miRNAs originating from this prostate-specific fraction of nsEVs more accurately reflects the process of prostate cancer development and has a greater diagnostic potential. Objective: to develop the method of miRNA isolation from the prostate-specific fraction of plasma nsEVs and to evaluate its performance characteristics.Materials and methods. Prostate-specific membrane antigen (PSMA) was used as a prostate-specific marker of nsEVs. The total population of plasma nsEVs was isolated using a two-phase polymer system. To isolate PSMA-positive (PSMA⁽⁺⁾) nsEVs, we used superparamagnetic particles with PSMA-binding DNA aptamer immobilized on their surface. The efficacy of PSMA⁽⁺⁾ nsEV isolation was assessed using flow cytometry and dot-blotting. RNA from nsEVs was isolated using proteolysis; miRNA analysis was performed using reverse transcription polymerase chain reaction. Plasma samples collected from patients with prostate cancer (n = 33) and healthy donors (controls) (n = 30) were used to evaluate the diagnostic parameters of the method.Results. We developed the method of PSMA⁽⁺⁾ nsEV isolation from plasma and estimated its performance characteristics. We found that measurement of potential miRNA markers in PSMA⁽⁺⁾ nsEVs was more effective than its measurement in the entire nsEV population and could distinguish between patients with prostate cancer and controls.Conclusion. The new technique of PSMA⁽⁺⁾ nsEV isolation can be used for the development of novel diagnostic methods for the diagnosis of prostate cancer.