Here, we describe a lateral flow biosensor capable of detecting H1N1 virus by integrating duplex-specific nuclease (DSN) and a novel isothermal amplification, two probe isothermal amplification (TPIA). Probe A and probe B of TPIA form DNAs of varying lengths by repeated polymerization and displacement of each other. Probe A extends into single-stranded DNA (ssDNA) of varying lengths containing multiple hairpin structures. Probe B extends into double-stranded DNA hairpins of different lengths. Both the 5´ end of the single strand DNA (ssDNA) probe A and probe B were joined by a ssDNA tail via a C3 spacer. These tails were designed to be complementary to capture sequence of probe C (5 ´end modifies fluorescein [FAM]) immobilized on magnetic beads. Cyclical DSN cleavage of probe C was triggered by target H1N1 RNA to release a capture sequence to capture TPIA product DNA with multiple biotins. The TPIA product/capture sequence complex of FAM can be captured by anti-FAM in the test strip test area, and the accumulation of gold nanoparticles causes a red band to appear in the test area. The limit of detection of specific RNA was as low as 829 fM with a linear range from 1 pM to 100 nM. This visual detection system is suitable for influenza A H1N1 virus point-of-care diagnosis in non-specialist personnel and low-resource settings.