Objective To observe the sterilization effects of hydrogen peroxide vapor on hard surface and air in laboratory animals barrier environment. Methods BIOQUELL Z sterilizer continuously sprayed 30% hydrogen peroxide vapor by 6 mL/m3、9 mL/m3 and 12 mL/m3 for 60 min at different positions in laboratory animal barrier environment. The numbers of falling bacteria and bacteria on the surface in barrier environment were detected. Rusults The falling bacteria in second changing rooms, clean corridor, storage room, dirt corridor and buffer room were killed completely by amount of 6 mL/m3 H2O2, and the falling bacteria in animal feeding room were killed completely by amount of 9 mL/m3 H2O2, but the bacteria on the surface in barrier environment were not completely killed. The bacteria on the surface in barrier environment were completely killed by amount of 12 mL/m3 H2O2. Conclusion Hydrogen peroxide vapor has very good sterilization effects on laboratory animal barrier environment and housing facilities.