Influenza is an acute respiratory infection disease caused by the influenza virus. At present, due to the high mutation rate of influenza virus, it is difficult for the existing antiviral drugs to play an effective antiviral effect continually, so it is urgent to develop a new anti-influenza drug. Recently, more and more studies have been conducted on the antiviral activity of Astragalus membranaceus, but the specific antiviral mechanism of this traditional Chinese medicine is not clear. In this study, the results proved that the Astragalus membranaceus injection showed obvious anti-influenza virus activity. It could improve the survival rate of Raw264.7 cells which were infected with influenza virus, while it improved the blocking effect of influenza virus on cell cycle after infection, increased the SOD activity, and reduced the MDA content. At the same time, the innate immunity was affected by regulating the expression of TLR3, TAK1, TBK1, IRF3, and IFN-β in the TLR3-mediated signaling pathway, thus exerting its antiviral effect in vitro.