The objective of our study was to develop and evaluate a TaqMan real-time RT-PCR (RRT-PCR) assay for universal detection of influenza A (IA) viruses. The primers and LNA-modified octanucleotide probe were selected to correspond to extremely conserved regions of the membrane protein (MP) segment identified by a comprehensive bioinformatics analysis including 10,405 IA viruses MP sequences, i.e., all of the sequences of the Influenza Virus Sequence database collected as of August 20, 2009. The RRT-PCR has a detection limit of approximately five copies of target RNA/reaction and excellent reaction parameters tested in four IA viruses reference laboratories. The inclusivity of the assay was estimated at both the bioinformatic and the experimental level. Our results predicted that this RRT-PCR assay was able to detect 99.5% of known human IA virus strains, 99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian strains, 98.89% of swine strains, 98.15% of equine strains, and 100% of influenza A viruses of other origin.