Polycythemia vera (PV) is a myeloproliferative disease characterized by hemoglobin/hematocrit levels above 16.5 g/dL or 49% in men, and 16 g/dL or 48% in women and accompanied by JAK2 mutations. It is aimed to prevent false negative and false positive results with simultaneous serum erythropoietin (EPO) measurements. The importance of sensitivity and specificity of low EPO level for PV is known; determining a more sensitive cut-off may reduce the need for further analyzes and invasive procedures. In our study, we aimed to reveal the sensitivity and specificity of serum EPO level in patients diagnosed with PV and to determine a new cut-off value that would show higher diagnostic performance instead of low serum EPO level. In this study, patients who were examined because of polycythemia between 01.01.2018–01.01.2022 at the hematology clinics of 5 different centers from xxx were included. A total of 468 patients (90 females, 378 males) were included in the study. One hundred seventy-five (37.4%) patients met the criteria for PV. In the ROC analysis performed to reveal the effectiveness of serum EPO level for predicting the diagnosis of PV, the sensitivity for ≤ 4.68 cut-off value was 89%, specificity 92%, AUC = 0.934 (95% CI: 0.91–0.96, p < 0.05), positive predictive value 87% and negative predictive value was 93.1%. Serum EPO level is an effective and easy method for predicting the diagnosis of PV, and it would be more predictive for the new cut-off level in this study.