A universal primer set was developed to amplify the full-length genome of all influenza A viruses. The primers were designed based on the conserved terminal nucleotides of the viral RNA segments, which are common to all influenza A viruses but unique for each segment. The primers contain influenza-specific nucleotides at the 3'-end and non-influenza nucleotides at the 5'-end to ensure specificity. This primer set allows the amplification of all eight segments of influenza A viruses, including the neuraminidase genes for N3, N6, N7, and N9 subtypes, which were previously difficult to amplify. The primers were optimized to ensure the amplification of these genes. The resulting primer set is suitable for generating full-length cDNAs, subtyping viruses, sequencing their DNA, and constructing expression plasmids for reverse genetics systems. The method was tested with various influenza A viruses and showed successful amplification of all eight segments. The primers were also used for the amplification of the hemagglutinin and neuraminidase genes, with specific primers designed for N3, N6, N7, and N9 subtypes. The universal primers allow for the amplification of all influenza A virus subtypes without prior subtype determination. The method is efficient, reliable, and suitable for rapid identification and characterization of all 15 HA and 9 NA subtypes. The primers were also optimized for cloning into plasmids, facilitating the generation of recombinant infectious viruses. This universal primer set is a powerful tool for studying influenza A viruses and has potential applications in vaccine development and pandemic preparedness.A universal primer set was developed to amplify the full-length genome of all influenza A viruses. The primers were designed based on the conserved terminal nucleotides of the viral RNA segments, which are common to all influenza A viruses but unique for each segment. The primers contain influenza-specific nucleotides at the 3'-end and non-influenza nucleotides at the 5'-end to ensure specificity. This primer set allows the amplification of all eight segments of influenza A viruses, including the neuraminidase genes for N3, N6, N7, and N9 subtypes, which were previously difficult to amplify. The primers were optimized to ensure the amplification of these genes. The resulting primer set is suitable for generating full-length cDNAs, subtyping viruses, sequencing their DNA, and constructing expression plasmids for reverse genetics systems. The method was tested with various influenza A viruses and showed successful amplification of all eight segments. The primers were also used for the amplification of the hemagglutinin and neuraminidase genes, with specific primers designed for N3, N6, N7, and N9 subtypes. The universal primers allow for the amplification of all influenza A virus subtypes without prior subtype determination. The method is efficient, reliable, and suitable for rapid identification and characterization of all 15 HA and 9 NA subtypes. The primers were also optimized for cloning into plasmids, facilitating the generation of recombinant infectious viruses. This universal primer set is a powerful tool for studying influenza A viruses and has potential applications in vaccine development and pandemic preparedness.