The rapid and sensitive detection of H5N1, a highly pathogenic avian influenza virus, is crucial for controlling its spread and minimizing its impact on public health. In this study, we developed a novel biosensor based on strand displacement amplification (SDA) coupled with CRISPR/Cas12a for highly sensitive detection of H5N1 DNA. The biosensor utilizes a combination of a three-way junction structure, composed of three hairpins (H1, H2, H3), to initiate amplification through SDA, resulting in the production of numerous activators. These activators then trigger CRISPR/Cas12a´s collateral cleavage activity, which generates a detectable fluorescence signal. The biosensor demonstrated a linear detection range from 100 fM to 800 pM, with a detection limit as low as 72.87 fM. The optimized biosensor exhibited excellent sensitivity, high specificity, and a broad dynamic range, making it a promising tool for the early detection of H5N1 DNA in complex biological samples. Additionally, the use of CRISPR/Cas12a´s trans-cleavage activity significantly improved signal amplification and specificity, allowing for more reliable detection compared to traditional methods. The results highlight the advantages of the integrated SDA and CRISPR/Cas12a approach, which addresses the limitations of conventional detection methods, such as low sensitivity, lengthy analysis times, and high costs. The biosensor´s ability to perform well in complex sample matrices demonstrates its potential for point-of-care diagnostics, especially in resource-limited settings. Future applications of this technology could extend to the detection of other pathogens, offering a versatile and adaptable platform for disease surveillance and management.