During the last few years, several cases of West Nile virus (WNV) infection in humans have been reported in Tunisia. However, detailed information on WNV infection in wild birds, the primary amplifying host of WNV, are lacking. In this work, we investigated the exposure of wild sparrows (hybrid Passer domesticus x hispaniolensis) living in two oases in southern Tunisia (Gabes and Kebili oases) to WNV, through the detection of WNV-specific antibodies by using ELISA and microneutralization tests. In total, 208 birds were sampled (54 from Kebili, 154 from Gabes). Anti-WNV antibodies were detected in two birds, corresponding to an overall seroprevalence of 1%. There was no significant difference between the two sampled populations [1.85% (1/54) in Kebili, 0.65% (1/154) in Gabes]. These data provide indirect evidence of the exposure of resident sparrows in southern Tunisia to WNV.