The animal laboratory needs to be fumigated and disinfected thoroughly before starting using in order to ensure the cleanness of the room. Ventilation system fumigation provides the fumigation gas generating equipment between air supply pipe and air discharge pipe to promote the fumigation gas diffusion through indoor air circulation. In order to study the influencing factors of gas concentration distribution in the fumigation process of the ventilation system, icem modeling and fluent software were used to carry out the transient simulation of air distribution in different conditions under different times，ventilation times，air supply modes and temperature distribution conditions, and to predict and analyze the distribution law of fumigation gas concentration and humidity. It is concluded that the minimum ventilation rate to quickly reach the sterilization degree decreases with time, the change of the air supply mode can reduce the vortex near the test bench, and the indoor temperature decreases with the height to facilitate the rapid diffusion of fumigation gas, so as to control the fumigation more effectively, which is of great significance to guide the safety of animal experiment.