Background Staff in tertiary hospitals are a high-risk group for occupational burnout. Timely identification and precise intervention are crucial for improving healthcare service quality. However, comparative studies on perceived stress and occupational burnout among hospital staff in different positions are lacking. Objective To describe the status of perceived stress and occupational burnout among hospital staff in different positions and compare the differences, explore the relationship between perceived stress and occupational burnout, and identify the influencing factors of occupational burnout. Methods In May 2022, 1526 hospital staff members were selected by convenience sampling from six tertiary hospitals in Guangzhou. Data were collected using a general information questionnaire, the Chinese Perceived Stress Scale (CPSS), and the Maslach Burnout Inventory-Human Services Survey (MBI-HSS) to assess demographic characteristics, perceived stress, and occupational burnout. Chi-square test, one-way ANOVA, and Kruskal-Wallis H test were used to compare differences in perceived stress and occupational burnout among staff in different positions. Pearson correlation analysis was used to explore possible correlation between perceived stress and occupational burnout. Multiple linear regression was conducted to identify influencing factors of occupational burnout. Results A total of 1388 valid questionnaires were collected, with an effective response rate of 90.96%. Among them, 437 nurses (57.0%), 273 physicians (58.6%), 24 pharmacists (57.1%), and 62 administrators (54.9%) reported positive perceived health risk stress (P=0.892). Additionally, 392 nurses (51.1%), 213 physicians (45.7%), 19 pharmacists (45.2%), and 53 administrators (46.9%) reported positive occupational burnout (P=0.005). Perceived stress was positively correlated with occupational burnout among nurses, physicians, pharmacists, and administrators (r=0.5973, 0.5687, 0.5464, and 0.5276, respectively; P＜0.01). The multiple linear regression identified perceived stress (b=1.4587, P＜0.001), job satisfaction (b=−7.4195, P＜0.001), self-rated health (b=−2.0428, P＜0.001), working years (b=−0.1135, P=0.016), and being a nurse (compared with an administrators) (b=3.2435, P=0.045) as significant factors for occupational burnout (P<0.001). Conclusion Perceived health risk stress and occupational burnout are common among hospital staff, with nurses experiencing significantly higher levels of occupational burnout compared to other positions. Hospital managers should pay more attention to staff with higher perceived stress, lower job satisfaction, poorer self-rated health, and shorter working years, particularly nurses.