Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative disorders, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, proteomic composition of these vesicles in PV patients has not been investigated before. In this work, we examined proteomic composition of serum exosomes of PV patients in comparison to healthy controls. We processed exosome-enriched serum samples using Multiple Enzyme Filter Aided Sample Preparation approach (MED-FASP), conducted LC-MS/MS measurements on a Q-Exactive HF-X mass spectrometer, and quantitatively analyzed the absolute concentrations of identified proteins by Total Protein Approach (TPA). 38 proteins were present at statistically significant different concentrations between PV patients’ study group and healthy controls’ group. The main protein components deregulated in PV were primarily related with excessive amounts of cells (TFRC, SELL, GP5), increased platelet activation (SERPINE1, MMRN1), elevated immune and inflammatory response (HPSE, CAMP, LYZ, SELL, LTF), and high concentrations of procoagulant and angiogenetic agents (ANG, HPSE). Our study provides the first quantitative analysis of the serum exosomes’ proteome in PV patients. Acquired knowledge can be beneficial to understanding of the mechanism of PV disease and further development of diagnostic or therapeutic procedures.