Background: West Nile virus is an infection that is most commonly caused by infected mosquito bites, however, blood transfusions, organ transplants, breast feeding, pregnant mother-to-the-fetus transmission, and occupational transmission among laboratory and medical staff are also the less common routes of infection. Given the endemic nature of this virus in the Middle East, the aim of this study was to investigate the presence of this virus in regular blood donors, as the reliable source of blood supply needed for patients in hospitals. Methods: In this descriptive-analytical study, venous blood samples were collected from 259 regular blood donors referred to the Blood Transfusion Organization of Kurdistan. After separating blood serum, the amount of IgM and IgG antibodies against West Nile virus was measured via ELISA test. Results: Concerning antibodies, IgG and IgM against West Nile virus were positive in 14 patients (5.4%) and 3 patients (1.2%), respectively. Seropositive IgG levels were observed in 11 patients over the age of 40 (12.5%) but only in 3 patients under 40 years of age (1.8%). The difference was statistically significant (OR = 7.95; 95% CI: 2.16–29.32; p < 0.01). Conclusion: Given the value of blood and blood products obtained from regular blood donors for therapeutic purposes and the significant prevalence of the virus and considering the presence of cases with positive IgM, it seems necessary to screen blood donors in blood transfusion centers in the western parts of Iran.