Background <p>Avian influenza viruses (AIVs), particularly the H7N9 subtype, pose a persistent threat to global health security, having caused 1,687 human infections and 615 deaths to date. The significant socioeconomic impact and pandemic potential of H7N9 underscore the urgent need for rapid, reliable diagnostic tools to facilitate early intervention and control measures.</p> Methods <p>This study conducted on 2025 developed and optimized a time-resolved fluorescence microsphere immunochromatographic strip (TRFICS) assay for the rapid detection of H7 AIVs. The assay was constructed using two specific monoclonal antibodies, 2H9 and 1H9. Comprehensive evaluations were conducted to assess the assay’s sensitivity, specificity, reproducibility, and stability. Furthermore, clinical utility was validated by comparing TRFICS results with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) using field samples (collected from 2013 to 2025).</p> Results <p>The optimized TRFICS assay achieved limits of detection (LOD) of 2^–5 hemagglutination units (HAU) for H7N9 in allantoic fluid and 0.01 ng/mL for purified H7N9 hemagglutinin protein within 15&#xa0;min. The assay demonstrated 100% concordance with RT-qPCR results in field sample testing. High specificity was observed with no cross-reactivity against other prevalent pathogens. Additionally, the assay exhibited excellent repeatability (relative standard deviation &lt; 8%) and maintained stable performance under various storage conditions.</p> Conclusion <p>The developed TRFICS assay represents a robust, sensitive, and rapid platform for the on-site identification of H7 AIVs. Its superior performance characteristics make it a valuable tool for effective surveillance and timely response to potential H7 AIV pandemics.</p>