Background: A major obstacle to successful coronavirus disease (COVID-19) vaccine rollout is vaccine hesitancy. Acceptance of and preferences for COVID-19 vaccination among healthcare workers (HCWs) is critical, because they are a key target group for vaccination programs, and they are also highly influential to vaccine uptake in the wider population. This study sought to comparatively determine the acceptance of and preference for COVID-19 vaccination among HCWs and the general population. Methods: An Internet-based, region-stratified discrete-choice experiment was conducted among 352 HCWs and 189 general population respondents recruited in March 2020 from 26 Chinese provinces. We accessed knowledge of disease, attitude towards and acceptance of COVID-19 vaccination. Several attributes (related to COVID-19 disease, COVID-19 vaccination and one social acceptance) were identified as key determinants of vaccine acceptance. Results: HCWs had a more in-depth understanding of COVID-19 and showed a higher willingness to accept COVID-19 vaccines with lower effectiveness and/or more severe adverse effects compared to the general population. 76.4% of HCWs (vs 72.5% of the general population) expressed willingness to receive vaccination (χ ² =2.904, p =0.234). High levels of willingness to accept influenza (65.3%) and pneumococcal (55.7%) vaccination were also seen in HCWs. Future COVID-19 disease incidence (OR: 4.367, 95% CI 3.721–5.126), decisions about vaccination among social contacts of respondents (OR 0.398, 95% CI 0.339–0.467), and infection risk >30% (OR 2.706, 95% CI 1.776–2.425) significantly increased the probability of vaccination acceptance in HCWs. Conclusion: Multi-component interventions to address the key determinants of hesitancy in both HCWs and in the general population should be considered for COVID-19 vaccination programs.