We have developed a rapid, duplexed microsphere-based immunoassay for the characterization of influenza virus types that has the potential to overcome many of the limitations of current detection methods. The assay uses microspheres of two sizes, each coupled to an influenza type A- or type B-specific monoclonal antibody (MAb), to capture influenza viruses in the sample. A cocktail of fluorescently labeled, influenza-specific polyclonal antibodies then binds the captured viruses. The sandwich complexes are measured using a multiparameter flow cytometer. The assay can distinguish between influenza types A and B in a single reaction with good reproducibility and high sensitivity. Detection sensitivity is much higher than that of commercially available influenza diagnosis quick kits: the FLU OIA (Thermo Biostar) kit and the Directigen Flu A+B kit (Becton Dickinson). The multiplexing capabilities of the current assay, which are not possible with enzyme-linked immunosorbent assay (ELISA) and the commercially available kits, reduce sample handling and consume fewer costly reagents. This assay represents a more efficient and sensitive method of characterizing influenza types. With inclusion of influenza subtype-specific antibodies as capture antibodies, this microsphere-based immunoassay can be expanded to differentiate among influenza types and subtypes in a single reaction to improve world-wide influenza surveillance.