The coronavirus disease 2019 (COVID-19) pandemic results in a profound physical and mental burden on healthcare professionals. This study aims to evaluate burnout status and mood disorder of healthcare workers during this period. An online questionnaire was voluntarily answered by eligible adult employees in a COVID-19 specialized medical center. The major analysis included the burnout status and mood disorder. Factors related to more severe mood disorder were also identified. A total of 2029 participants completed the questionnaire. There were 901 (44.4%) and 923 (45.5%) participants with moderate to severe personal and work-related burnout status, respectively. Nurses working in the emergency room (ER), intensive care unit (ICU)/isolation wards, and general wards, as well as those with patient contact, had significantly higher scores for personal burnout, work-related burnout, and mood disorder. This investigation identified 271 participants (13.35%) with moderate to severe mood disorder linked to higher personal/work-related burnout scores and a more advanced burnout status. Univariate analysis revealed that nurses working in the ER and ICU/isolation wards were associated with moderate to severe mood disorder risk factors. Multivariate analysis demonstrated that working in the ER (OR, 2.81; 95% CI, 1.14–6.90) was the only independent risk factor. More rest, perquisites, and an adequate supply of personal protection equipment were the most desired assistance from the hospital. Compared with the non-pandemic period (2019), employees working during the COVID-19 pandemic (2020) have higher burnout scores and percentages of severe burnout. In conclusion, this study suggests that the COVID-19 pandemic has had an adverse impact on healthcare professionals. Adequate measures should be adopted as early as possible to support the healthcare system.