Here, we developed a novel single primer-amplification (SPA) method for detection of nucleic acids that requires only two primers and one enzyme. The primer was extended into long ssDNA products with tandem repeats of varying lengths via continuous polymerization and dissociation from the template. The isothermal SPA was integrated with a lateral flow assay (LFA) to visually detect RNA of the H1N1 influenza virus. In the presence of the target RNA, a FAM probe/target RNA/long ssDNA (SPA product) sandwich complex was integrated into the sample pad and labeled with gold nanoparticles onto the test strip, which were captured by the T line of the LFA test strip. The limit of detection of H1N1 RNA was as low as 18 pM. The SPA-LFA produced reliable and specific results in less than 1?h. The proposed biosensor is a simple and sensitive in vitro diagnostic tool for detection of the H1N1 virus and is suitable for point-of-care diagnosis by non-specialist personnel in low-resource settings.