Animal Biosafety Level 3 Laboratory (ABSL-3), as a basic support platform for the national biosafety system for emerging and re-emerging infectious diseases, provides a crucial infrastructure for the study of the biological characteristics of highly pathological pathogenic microorganisms, prevention and treatment means.The ABSL-3 laboratory also provides an important support for the university “industry-academia-research” combination. The normal operation of the ABSL-3 laboratory and the smooth development of scientific research work can not be separated from the careful planning and effective implementation of emergency preparedness work. In addition, the reasonable design and layout of the laboratory is also a key factor to avoid and reduce accidents. This paper combines Yangzhou University ABSL-3 construction and management practices, starting from the preparation of the emergency plan on the laboratory construction and layout, daily operation and sewage treatment, terminal disinfection of specific measures, and emergency disposal and other aspects of attention to be introduced, in order to provide a useful reference for the future operation of various pathogenic microorganisms biosafety laboratories.