A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the Avian Influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for the detection. The outstanding feature of this assay is its capability to designate both the HA and NA of an AIV sample from a single reaction. A panel of 77 influenza strains was tested representing the entire assortment of the two antigens. 100% (77/77) of the samples tested were identified as AIV and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.