Background Brucellosis is a zoonotic disease with economic and public health significance in developing countries that rely on livestock production including Ethiopia. This study intended to establish the seroprevalence and associated risk factors of ovine brucellosis. Methods A cross-sectional study was carried out on seroepidemiology of ovine brucellosis from January 2017 to June 2020 G.C in five districts of South Omo zone, Southern Ethiopia. A total of 1536 sera samples were collected from sheep and serially tested using modified Rose Bengal plate test, competitive enzyme-linked immunosorbent assay, and complement fixation test to detect antibodies against natural infection by Brucella species. A structured questionnaire was used to collect data from individual animals, and flocks for the analysis of the association between expounding and outcome variables. Data were analyzed using STATA version 14.0 and potential risk factors for seropositivity of brucellosis were analyzed using logistic regression. Results The study discovered an overall 5.40% (95% CI: 6.34, 14.25) and 39.74% (95% CI: 6.50, 6.97) seroprevalence of ovine brucellosis at individual and flock level, respectively, by a confirmatory test. Age groups, sex, flock size, district, history of abortion, and body condition were significantly associated risk factors with Brucella seropositivity (p < 0.05). Conclusion To conclude, the prevalence of ovine brucellosis in the South Omo Zone was relatively high which needs integrated intervention approaches in place to curb the spread of the disease.