Influenza virus infections, particularly those caused by the Avian influenza virus, result in millions of deaths and substantial economic losses annually. Early diagnosis of the pathogen during the initial stages of an outbreak is pivotal for effective outbreak control. This study presents the development of an Exo III-mediated high sensitivity dual signal amplification (HSDSA) platform for detecting the H5N1 gene sequence. The detection mechanism is initiated by the hybridization of the target H5N1 DNA with hairpin DNA (Hp DNA) to form double-stranded structure. Hp DNA in this structure could be recognized and partial hydrolyzed by Exo III, releasing target DNA and free DNA. On one side, the target DNA enabled hybridize with other Hp DNA for circulate freely, achieving the first round of amplification. On the other hand, free DNA can hybridize with the DNA structure labeled on the gold nanoparticles, transforming the originally 3-terminal protruding double-stranded structure into a 3-terminal concave Exo III substrate structure. After triggering the Exo III recognition, the long chains (SH DNA) within dsDNA are hydrolyzed, and free DNA is released again to enter the next round of triggering for the second round of amplification. Meanwhile, another DNA labeled with carboxyfluorescein (FAM DNA) released from the hydrolyzed SH DNA, causing the fluorophore to detach from the AuNPs, the fluorescence signal is restored. This strategy significantly enhances detection sensitivity through double cyclic signal amplification mediated by Exo III, with a linear range from 0.08?nM to 15?nM and a detection limit of 54 pM for H5N1 DNA. The fluorescence sensor demonstrates robust applicability in detecting H5N1 in human serum samples, with recovery rates ranging from 98.75% to 105.83%. These results underscore the potential of the HSDSA platform for low-abundance molecular detection, disease diagnosis, and biomedicine applications.