Neuraminidase (NA) is one of the key enzymes responsible for bacterial infection and pathogenesis. This study aimed to gain deeper insights into the inhibitory effects of flavone-glucosides (1-9) isolated from barley sprouts (BS) on neuraminidase activity. The isolated compounds were identified as, lutonarin (1), saponarin (2), isoorientin (3), orientin (4), isovitexin (5), isoscoparin-7-O-[6-sinapoyl]-glucoside (6), isoscoparin-7-O-[6-feruloyl]-glucoside (7), isovitexin-7-O-[6-sinapoyl]-glucoside (8), and isovitexin-7-O-[6-feruloyl]-glucoside (9). Among them, compounds 1-5 exhibited neuraminidase-inhibitory activities in a dose-dependent manner, with IC50 values ranging from 20.1 to 32.7 μM, in a non-competitive inhibition mode according to kinetic studies. Moreover, the individual flavone-glucoside levels differed notably, in particular, lutonarin (1) and saponarin (2) were shown to be present in the greatest amounts, according to UPLC analysis. Consequently, our results suggest that BS may be utilized as an effective NA inhibitor in human health food, additives, and feed.