Background: Brucellosis is one of the most frequent contagious neglected bacterial diseases with serious veterinary and public health importance throughout the world. A cross-sectional study on animal brucellosis was conducted from October 2018 to July 2019 in southern and central Ethiopia with the objective of estimating seroprevalence and molecular detection. Blood samples were collected for serum extraction from a total of 4274 individual animals (cattle, small ruminants and camel) from 241 herds/flocks. Blood clots from seropositive animals were also tested for brucellosis via molecular techniques. Additionally, 13 vaginal swab samples were collected from animals with recent abortion history for bacterial isolation and molecular detection.Results: The extracted serum samples were tested using multispecies I-ELISA and the overall individual animal and herd level seroprevalence was 3.95% (169/4274) and 18.26% (44/241) respectively. The animal level seroprevalence at species level was 1.58%, 8.89%, 12.44% in bovine, small ruminants (sheep and goat) and camel, respectively. Herd level seroprevalence were 5.43%, 52.08%, 100% in bovine, small ruminant and camel, respectively. The animal and herd level seroprevalence of bovine from intensive and extensive systems was 1.1%, 2.87% and 9.2%, 50.00% respectively. Brucella species was isolated from 6/13 (46.15%) vaginal swab samples cultured on brucella selective agar, and shown to be B.melitensis using Real-Time PCR. All of the blood clots from seropositive animals were negative for the presence of Brucellaspp with PCR.Conclusions: In the regions sampled animals in all three categories – cattle, sheep/goats, and camels – mostly had low seroprevalence rates for brucellosis, without great differences from previous serosurveys done in Ethiopia. However, seropositivity for camels was higher than what has been reported previously. Also, there was a notable difference in this study in cattle seroprevalence when comparing extensive with intensive systems, with the extensive system being much higher.