Background: Healthcare workers are often exposed to stressful working conditions at work which affect their quality of life. The study investigated the relationship between psychosocial risk factors, stress, burnout, and quality of life among primary healthcare workers in general medical practice in Qingyuan and Chaozhou cities in Guangdong province. Method: The cross-sectional study was conducted in 108 primary health facilities including 36 community health centers (CHCs) across two developing cities in Guangdong province. A total of 873 healthcare workers completed the questionnaires. Quality of life was evaluated using The World Health Organization Quality of Life Questionnaire (WHOQOL-BREF) and psychological risk factors were evaluated by the Copenhagen Psychosocial Questionnaire (COPSOQ). General quality of life and the quality of life domains were transformed into a score range from minimum 0 to 100 maximum. Higher scores indicated better quality of life and vice versa. Significant associations were verified using multiple regression analysis. Results: Poor quality of life was observed in 74.6% of healthcare workers surveyed. General poor quality of life was significantly higher among workers who reported higher burnout (Beta = − 0.331, p < 0.001). In addition, workers with high levels of burnout, unmarried workers and female workers had a higher possibility of physical health. A greater risk of poor psychological health was observed among workers with high burnout, poor sense of community and those with lower educational levels. Workers who lacked social support, those with fewer possibilities for development had increased probability of poor quality of life in the social domain. Poor quality of life in the environmental domain was observed among workers who were dissatisfied with their jobs and workers with low salaries. Conclusions: Primary healthcare workers in developing cities in China have a highly demanding and strained working environment and poor quality of life. Reducing job stress and improving work conditions may ultimately improve the well-being of primary healthcare workers.