Rift Valley fever (RVF) is a re-emerging zoonotic disease of domestic ruminants and humans. While neighbouring countries have reported outbreaks of RVF, Ghana has not yet identified any cases. The aim of this study was to determine whether RVF virus (RVFV) was circulating in livestock and herders in the southern part of Ghana, to estimate its seroprevalence, and to identify associated risk factors. The study surveyed 165 livestock farms randomly selected from two districts in southern Ghana. Serum samples of 253 goats, 246 sheep, 220 cattle, and 157 herdsmen were tested to detect IgG and IgM antibodies against RVFV. The overall seroprevalence of anti-RVF antibodies in livestock was 13.1% and 30.9% of farms had RVFV seropositive animals. The species-specific prevalence was 24.1% in cattle, 8.5% in sheep, and 7.9% in goats. A RVFV IgG seroprevalence of 17.8% was found among the ruminant herders, with 8.3% of all herders being IgM positive. RVFV was shown, for the first time, to have been circulating in southern Ghana, with evidence of a recent outbreak in Kwahu East; however, it was clinically undetected despite significant recent human exposure. A One Health approach is recommended to better understand RVF epidemiology and socio-economic impact in Ghana.