Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.