Background: Reports of SARS-CoV-2 in animals have implicated human sources. Similarities in SARS-CoV-2 cell receptors between humans and animals suggest possible cross-transmission of COVID-19. Using a One-Health approach, we assessed exposure to SARS-CoV-2 in humans with no history of confirmed infection, selected domestic animals in their homesteads, and in wild animals in Uganda. Methods: A cross-sectional study was done in two districts where community COVID cases were first reported: and at Uganda Wildlife Education Centre (UWEC). Thirty-six humans and 48 domestic animals per district; and 14 animals at UWEC were sampled. Blood and nasopharyngeal or oropharyngeal samples were analysed for SARS-CoV-2 antibodies, viral antigens, and genome. Interviewer-administered questionnaire on socio-demographics, travel history, disease symptoms and knowledge on COVID-19 was conducted, data was analysed with STATA 16.0. Results: One hundred forty participants were interviewed, 77% were below 49 years old, 56% had only primary education, 50% were catholics; and 70% subsistence farmers. COVID-19-related information was majorly from radios (89%), families and or friends (55%). Over 60% were aware of at least two COVID-19 symptoms. In humans, sero-prevalence was 31.9%, 4.2% and 13.9% for IgG, IgM, and COVID-19 antigen, respectively. IgG sero-prevalence was significantly higher in females (47.2%) than males (16.7%) with p=0.005, and similar trends were noted for IgM and antigens. In domestic animals, caprine had the highest IgG seropositivity, followed by bovine, avian and lastly canine at 70.6%, 50.0%, 42.9% and 38.5%, respectively. IgM was detected in one dog. At UWEC, five animals had both IgG and IgM whereas two had only IgG. All human and animal samples were RT-PCR negative. Conclusion: A high sero-prevalence of IgG, indicates previous exposure. Ascertaining cross-species transmission at human-animal interface was unproven due to negative RT-PCR results, hence isolation and characterisation of virus were not done.