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The outbreak of COVID-19 has caused increasing public attention to laboratory-acquired infections (LAIs), especially for a mobile Bio-Safety Level 4 Lab (BSL-4) with high potential of exposure. In this paper, the distribution and removal mechanism of bioaerosols in the biosafety laboratory were studied. A simulation model of airflow distribution in the opening and closing state of air-tight door was established and verified. The results showed that the airflow entrainment velocity during the opening of the door was approximately 0.12 m/s. It increased the probability of vortex generation in the laboratory. The deposition rate of particles was doubled when the air-tight door opening is compared with air-tight door closing. Besides, nearly 80% of the particles deposited on the surface of the wall and ceiling, increasing the possibility of LAIs. The findings of this paper could provide new scientific methods for high-level biosafety laboratories to avoid cross-infection. Moreover, future work regarding air-tight door rotation speed regulation and control should be emphasized.Keywords Laboratory-acquired infections . Mobile Bio-Safety Level 4 Lab . Removal mechanism . Airflow distribution . Bioaerosols . Entrainment velocity Highlights• This study was conducted based on a mobile BSL-4 laboratory.• The mechanism of bioaerosol diffusion between multi-region was analyzed.• Entrainment velocity altered the locations of high concentration zones of bioaerosols.• Opening or closing of airtight doors raises the possibility of cross-infection.• The mode of interaction between airflow and bioaerosols was explored.