For the highly pathogenic H5N1 avian influenza virus, the construction of highly sensitive biosensors to detect the virus, combined with intelligent packaging for real-time detection, is of great significance for the prevention and control of avian influenza. Based on HCR signal amplification strategy and using AgNCs as fluorescence signal group, a labeled “turn on” fluorescent biosensor was constructed to detect H5N1 gene sequence representing H5N1 virus. The sensor used H5N1 DNA as a trigger to trigger the HCR process, which caused AgNCs to produce strong fluorescence signal changes. The research has shown that the sensor had a good response signal when the H5N1 DNA concentration was within 0.2~800.0 nmol/L, and the fluorescence intensity was linearly correlated with the H5N1 DNA concentration between 0.2~200.0 nmol/L. The linear equation was y = 10.982C + 567.435 (R2 = 0.992 73), and the detection limit was 176 pmol/L. The nucleic acid sensing system is universal, and can realize specific sensitive detection of different targets by simply adjusting the target sequence. This research is expected to provide ideas for the design of a universal sensing platform for highly sensitive analysis of avian influenza virus markers.