Background Brucellosis is a worldwide zoonosis with significant impact on rural livelihoods and a potentially underestimated contributor to febrile illnesses. The aim of this study was to estimate the seroprevalence of brucellosis in humans and small ruminants in The Gambia. Methods The study was carried out in rural and urban areas. In 12 rural villages in Kiang West district, sera were collected from humans (n = 599) and small ruminants (n = 623) from the same compounds. From lactating small ruminants, milk samples and vaginal swabs were obtained. At the urban study sites, sera were collected from small ruminants (n = 500) from slaughterhouses and livestock markets. Information on possible risk factors for seropositivity was collected through questionnaires. Sera were screened for antibodies against Brucella spp. with the Rose Bengal Test, ELISA and Micro Agglutination Test (human sera only). PCR was performed on 10 percent of the milk samples and vaginal swabs from small ruminants. Results One human and 14 sheep sera were positive by the Rose Bengal Test. The rest were negative in all serological tests used. The PCR results were all negative.Conclusions The results suggest that brucellosis is currently not a generalized problem in humans or small ruminants in The Gambia.