Abstract: Objective　To monitor the sampling process of animal biosafety level 3 (ABSL-3) lab during Mycobacterium tuberculosis (MTB) animal model construction experiments, and provide basis for biosafety evaluation of ABSL-3 lab.   Methods　During the process of MTB model construction， air samples and facility surface samples were collected from the authorized ABSL-3 lab by China National Accreditation Service for Conformity Assessment (CNAS). The rapid molecule technology and traditional pathogenic method were used in samples′ specificity detection.  Results　General environmental microbes could be tested in biosafety level 3 (BSL-3) lab，but the core room was cleaner than preparing room most probably because of the negative air pressure and high cleaning frequency. The 70% alcohol can reduce general bacterium detection rate from 59.26% to 9.38%, from 6.59% to 1.52% in preparing room and from 10.99% to 0 in core room. The MTB aerosol contamination was not detected in ABSL-3 and biohazard safety equipment under dual negative pressure.  Conclusions　The appropriate negative air pressure and proper use of biosafety cabinet sterilization measures could help to avoid MTB contamination and ensure the biosafety of ABSL-3 lab.

Key words: Biosafety evaluation, Animal biosafety level 3 lab, Mycobacterium tuberculosis, Animal model