The current immediate detection of influenza A (H1N1) virus faces the dual challenges of insufficient sensitivity and the risk of amplification contamination. Here, we developed a novel lateral flow nucleic acid biosensor using pre-amplified dsDNA tag (PADT). Linearized M13 phage was preamplified to generate a 3.5-kbp dsDNA product containing a 20-nt overhang probe and heavy biotin tags, followed by the formation of FAM probe/target RNA/PADT complexes by a sandwich-type detection strategy.