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West Nile virus (WNV) is a globally significant mosquito-borne Flavivirus that causes West Nile disease (WND). In Libya, evidence of WNV circulation has been reported in humans but never in animals. The aim of this study was to determine the seroprevalence of WNV infection in horses and dogs in Libya. In total, 574 and 63 serum samples were collected from apparently healthy, unvaccinated horses and dogs, respectively, between 2016 and 2019. A commercially available competitive enzyme-linked immunosorbent assay (c-ELISA) kit was initially used to test the collected samples for the presence of WNV Ig-G antibodies. Positive and doubtful sera were also tested using a more specific virus neutralisation assay to confirm whether the ELISA-positive results were due to WNV or other Flavivirus antibodies. The seroprevalence of WNV IgG antibodies according to ELISA was 13.2% out of 574 of total horses' samples and 30.2% out of 63 of total dogs' samples. The virus neutralisation test (VNT) confirmed that 10.8% (62/574) and 27% (17/63) were positive for WNV-neutralising titres ranging from 1:10 to 1:640. Univariable analysis using chi-square tests was conducted to measure the statistical significance of the association between the hypothesized risk factors including city, sex, breed, and age group and were then analyzed using the subsequent multivariable logistic regression model for horse samples. Age group was found to be the only significant risk factor in this study. The results of the present study provide new evidence about WNV circulation in Libya. [ABSTRACT FROM AUTHOR]