Respiratory infections caused by influenza A viruses (IAVs) pose a global threat annually, leading to significant mortality in severe cases. IAVs are classified into various subtypes, and a combination of multiplex reverse transcription-multiple cross displacement amplification (mRT-MCDA) and lateral flow biosensor (LFB) has been developed to detect three subtypes (H1N1, H3N2, and H7N9) that have been frequently observed in recent years. This technology simultaneously reverse transcribes and amplifies two target genes, hemagglutinin (HA) and neuraminidase (NA), at a constant temperature of 63 °C in just 40?min. The final results can be read using a dual-channel LFB. In practice, the entire process—from sample collection, RNA extraction, and mRT-MCDA amplification to result interpretation—can be completed in less than 1?h. This method is named "multiplex reverse transcription-multiple cross displacement amplification-lateral flow biosensor" (IAVs-mRT-MCDA-LFB) technology. The IAVs-mRT-MCDA-LFB technology demonstrated higher sensitivity compared to conventional reverse transcription-polymerase chain reaction (RT-PCR) and showed no cross-reactivity with other genes present in the samples. Therefore, the IAVs-mRT-MCDA-LFB technique developed in this study is a highly valuable molecular diagnostic tool for detecting IAV-subtypes due to its simplicity, time efficiency, cost-effectiveness, and high specificity and sensitivity.