To study the activity of Waigan Qingre Jiedu decoction against respiratory virus in vitro. Methods:MDCK, HEL and A549 cells were cultured in vitro; influenza virus H1N1, adenovirus AV1 and respiratory synthetic virus RSV1 were inoculated respectively. Waigan Qingre Jiedu decoction was given to intervene, and Ribavirin was given as control. MTT method was used to detect the cytotoxicity of the drug and therapeutic index. Hemagglutination test and Cytopathic effect were used to study the effect against respiratory virus. Results:IC50 of Waigan Qingrejiedu decoction against H1N1 was 0.028 mg/mL. TI was 2.07. In the prevention administration experiment, hemagglutination test at the concentration of 0.01 mg/mL was negative, which indicates that the decoction could block H1N1 virus invasion and has preventive effect. In the therapy administration experiment, hemagglutination test at the concentration of 0.01 mg/mL was negative, which showed that the decoction had a therapeutic effect on the virus. In the Direct killing experiment, the decoction can reduce the virus titer by 1 gradient. Hemagglutination test against AV1 at the concentration of 0.005 mg/mL was negative, which showed that the decoction could inhibit AV1. The observation of Cytopathic effect under the microscope showed that the decoction could inhibit RSV1 in the concentration of 0.01 mg/mL. Conclusion:Waigan Qingre Jiedu decoction has high activity against H1N1, AV1 and RSV1, with the advantage of having high efficiency and low toxicity.