A new polyacetylenic glucoside together with three known compounds were isolated from the whole herb of Bidens pilosa L. The structure elucidation of these four compounds was employed by combining NMR and HR-MS data as 4-O-β-D-glucopyranosyloxy-1-hydroxy- 6-(E)-tetradecene-8,10,12-triyne (1), 3-O-β-D-glucopyranosyloxy-1-hydroxy-6-(E)-tetradec- ene-8,10,12-triyne (2), 2-O-β-D-glucopyranosyloxy-1-hydroxy-5-(E)-tetradecene-7,9,11-triy- ne (3) and icthyothereol acetate (4). Additionally, bioactivity study showed that these compounds have potential anti-inflammatory activity. This study has certain guiding significance for the development and utilisation of polyacetylene compounds in Bidens pilosa L.