Whole-genome sequencing (WGS) is essential for monitoring the genetic diversity of influenza A virus (IAV) across host species. We optimized a multisegment RT-PCR (mRT-PCR) protocol to enhance amplification of all eight IAV segments using modified RT and PCR conditions. Additionally, we introduced a dual-barcoding approach for the Oxford Nanopore platform, enabling high-throughput multiplexing without compromising sensitivity. The resulting workflow is robust, scalable, and effective for avian, swine, and human IAV samples, even at low viral loads. This approach strengthens genomic surveillance at the human-animal interface, supporting early detection, evolutionary monitoring, and rapid identification of IAV spillover events.