This cross-sectional study evaluated and quantified the possible association of psychological symptoms and health status ratings on the burnout of healthcare workers from a tertiary medical center. Demographic data were obtained through a questionnaire survey. We evaluated their psychological symptoms using a 5-item Brief Symptom Rating Scale (BSRS-5) and burnout was measured using the Chinese version of the Maslach Burnout Inventory–Health Services Survey. The study began in Nov. 2018 and ended in Nov. 2020. A total of 2813 participants (men = 296, 10.5%; women = 2517, 89.5%) completed the questionnaires between December 2018 and March 2019. The response rate and complete rate were 71.7% and 93.2%. The multivariate analysis showed that, as the BSRS-5 level added, the odds ratio (OR) of burnout increased (BSRS-5 scores 6–9, 10–14, and 15–20; OR = 1.83, 3.23, and 9.15, separately; p value < 0.05). Overall, men (≥ 30 years of age) and women staffs with BSRS-5 scores ≥ 6; women with longer working hours (more than 46 h/week), men and women (≥ 30 years of age) working night shifts, and poor health status ratings were highly associated with burnout. The findings highlight the importance of screening for the BSRS-5 scores and health status ratings level for healthcare professionals at high risk of burnout, especially men ≥ 30 years of age and women with stressful working conditions.