A new seco-neolignan glycoside, seco-dehydrodiconiferyl alcohol-4-O-β-D-glucopyranoside, together with eight known compounds, were obtained from the EtOH extract of the root bark of Ailanthus altissima. Their structures were elucidated based on the spectroscopic data. Three neolignan glycosides including 7,9,9'-trihydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan-4-O-β-D-glucopyranoside, sonchifolignan B and citrusin B exhibited moderate in vitro inhibitory effect on tobacco mosaic virus replication with IC₅₀ values 0.30, 0.35 and 0.26 mmol L⁻¹, respectively.