Elsholtzia rugulosa (Lamiaceae), a common Chinese herb, is widely used in the treatment of cold and fever. In order to elucidate the action mechanism and the active principles from the plant against anti-influenza virus, the influenza virus neuraminidase (NA) activity assay and in vitro antiviral activity assay were established, and the isolation of the active principles was guided by NA activity. Finally, 5 active constituents were obtained, namely apigenin (1), luteolin (2), apiin (3), galuteolin (4) and luteolin 3′-glucuronyl acid methyl ester (5), respectively. They all belong to the flavonoids. The in vitro antiviral assay using a cytopathic effect (CPE) reduction method showed that they all possessed anti-influenza virus activity. Among them, apigenin and luteolin exhibited the highest activities against influenza virus (H3N2) with IC50 values of 1.43 Mg/mL and 2.06 μg/mL, respectively. The structure-activity relationship (SAR) of these flavonoids with different chemical structures and their anti-influenza virus activities was addressed in this study.