Objective The staffs of biosafety level 3 laboratories (BSL-3) face with the stress of handling highly pathogenic microbs and special laboratory environment. The job stress may result in accidents in the laboratory as negative factor for the risk control. The research may provide support for the control of risk in biosafety laboratories. Methods In order to assess the job stress in the staff in BSL-3 laboratory, we modified "the Chinese simple job stress questionnaire" based on the theory of the JDC mode and ERI mode, and an investigation was carried out. The present study included the staffs (87 employees) from six BSL-3 laboratories located in five provinces (Shanghai, Zhejiang, Jiangsu, Fujian and Wuhan). Results Analysis of the data indicates that variables of age, working years, job duties, manipulating of animals, type of microorganisms and transmission route have a significant influence on the level of job stress in BSL-3 laboratory. Conclusion The BSL-3 laboratory staff in higher stress level have the characteristicses: 20-39 years old, short work years, regular staff, operating on air-borne microbiology, manipulating of animals and operating on one more microbiology.