We sought to investigate job stress among primary healthcare workers, to analyze the job stress among them in Jiangsu Province and provide evidence for exploring benign talent development mechanism in primary medical industry. A questionnaire was used to survey the state of job stress among 905 primary healthcare workers. The overall job stress score was 56.54, the doctors’ job stress score in township hospitals was higher than that of the doctors in urban community health service centers. Through the single factor analysis, the overall stress had significantly difference among gender, age, marital status, education background, technical class, type of unit, workload, working years, patients’ respect and social class. Furthermore, multivariate linear regression analysis results showed that social class, workload, working years, patients’ respect, education background and gender had significant impact on overall stress. We should pay more attention to the doctors in township hospitals, improve primary healthcare workers’ social class and focus on backbone in time to reduce job stress and improve work motivation.