Laboratories—General Requirements for Biosafety（GB 19489-2008） required “Exhaust high efficiency particulate air (HEPA) filter of Biosafety Level 3 Laboratory (BSL-3) should be sterilized in situ”. Exhaust HEPA filter is an important defensive line to ensure the biosafety of BSL-3 laboratory facilities and also the main line of defense to ensure the non-leakage of infectious aerosols generated in the experimental procedure. In situ disinfection of the HEPA filter is needed in order to protect the safety of personnel. This study designed and produced a cycle disinfection prototype (with the function of formalin volatilization, gas humidification and air circulation supply). The prototype with the disinfection interface of HEPA filter’s exhaust box were connected. And then the steam cycle of formalin was driven through the HEPA filter by air circulation supply system. Thus the in situ disinfection was realized. The results showed that formalin vapor concentration at room temperature was 16 mg/L, and the relative humidity was 70%. After 12-hour continuous fumigation cycle running through, Staphylococcus aureus, Mycobacterium smegmatis, Bacillus subtilis and Bacillus stearothermophilus were completely disinfected. As a consequence, the in situ disinfection of HEPA exhaust-air filter in both equipment and methodology is achieved.