Extracellular vesicles (EVs) are small membrane-bound sacs, identified in many body fluids of humans. Standard extracellular vesicle separation methods such as differential and ultracentrifugation are very expensive, not affordable in academic labs. So, the current research tried to isolate seminal plasma EVs using polyethylene glycol (PEG) precipitation process. Normospermia semen from “Milann - The Fertility Center” processed to isolate EVs by PEG method. Nanodrop spectrophotometer showed presence of EVs by indirectly measuring protein content of precipitated EVs. EVs isolated by PEG precipitation showed a wide size range from 30 to 1000 nm with Z average of 75.4 nm and a PI of 0.464, whereas ultracentrifuge sample showed size range of 60–1000 nm with Z average of 501.3 nm with a PI of 0.692. Edax analysis also showed good elemental pattern. Total RNA extraction from PEG EVs analysed with nanodrop spectrophotometer, showed presence of RNA content in varying concentrations obtained from different ratios in nanograms. Thus, the current study concludes that seminal plasma EVs isolated by PEG precipitation is simple, reproducible and non-sensitive to carry out at academic labs. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.