Background and Aim: West Nile fever (WNF) is a neurotropic, mosquito-borne disease affecting humans and domesticated animals, caused by a member of the genus Flavivirus. Over the last decades, this virus has been responsible for several cases of illness in humans and animals. The current epidemiological status of WNF in horses is insufficient, and in veterinarians, as an occupational hazard is unknown. This study aimed to investigate and determine the seroprevalence and risk factors for WNF in veterinarians and horses in Palestine. Materials and Methods: In this study, serum samples from 100 veterinarians and 87 horses were collected between August 2020 and September 2020 from different cities of Northern Palestine. West Nile virus (WNV) antibodies were detected using an enzyme-linked immunosorbent assay. Results: Our results showed that 60.9% of the horse serum samples were positive in all investigated cities. In horses, location is a risk factor for the seropositivity for WNF, whereas age, sex, breed, and intended use of the horses, were not associated with increased WNF seropositivity. In veterinarians, 23.0% of the serum samples were positive. Positive samples were detected in all locations, age groups, experience length, and work sectors. However, the seropositivity for WNF was not influenced by these variables. Conclusion: The results revealed that WNV circulates in most regions of Palestine. Our results will help determine the risk of infection in animals and humans and control WNV transmission. Surveillance studies on humans, vectors, and animals are needed to better define endemic areas.