Introduction We conducted a survey to investigate the uptake of COVID-19 vaccination among healthcare workers (HCWs) and older persons (≥ 50 years) in Uganda, groups at particular risk of infection and severe disease outcomes respectively. Methods The survey was conducted between May and August 2021, during the early phase of COVID-19 vaccine roll-out. All HCWs at Entebbe and Kisubi hospitals, Wakiso district (urban/peri-urban) and Villa Maria hospital, Kalungu district (rural), and non-healthcare worker (non-HCW) individuals enrolled in an ongoing older persons’ cohort study in Wakiso district, completed an interviewer-administered questionnaire on socio-demographics, pre-existing medical conditions, attitudes regarding COVID-19 vaccines, and vaccination uptake. Logistic regression was performed to investigate factors associated with uptake. Findings A total of 746 individuals, 597 HCWs and 149 non-HCW older persons, participated. Majority were aged ≤50 years (71.6%), female (63.1%), and had secondary school/other higher-level education (77.8%). COVID-19 uptake was 63.7%, overall; 95.2%, Entebbe Hospital; 75.4%, Kisubi Hospital; 49.5%, Villa Maria Hospital; and 20.8%, non-HCW. Among HCWs, health facility [Entebbe hospital (aOR 18.9, 95% CI 8.9-40.2), Kisubi hospital (aOR 5.2, 95% CI 3.0-9.0) all compared with Villa Maria], age >50 years (aOR 3.1, 95% CI 1.2-8.4) and positive attitude towards COVID-19 vaccines (aOR 5.0, 95% CI 2.1-11.8) were associated with high uptake, while female sex (aOR 0.5, 95% CI 0.3-0.9) was associated with low uptake. Among non-HCW older persons, absence of chronic infectious disease (aOR 4.3, 95% CI 1.1-16.3) good attitude towards COVID-19 vaccines (aOR 29.2, 95% CI 4.1-208.8) were associated with high uptake, while advanced age 70-79 years (aOR 0.1, 95% CI 0.01-0.97) was associated with low uptake. Conclusion COVID-19 vaccine uptake was lowest among rural-based HCWs and non-HCW older persons. Vaccination campaigns during a pandemic need to foster positive attitudes towards vaccines and employ strategies specifically designed to improve vaccine uptake among disadvantaged populations.