Rapid and simple methods for on-site detection of avian influenza virus (AIV) are essential for effective field surveillance. In this study, a microfluidic device-based non-competitive fluorescence polarization immunoassay (NC-FPIA) using an ATTO 647N-labeled hemagglutinin fragment of H5 subtype AIV (H5-AIV) as a tracer was developed for the quantification of H5-AIV in avian oropharyngeal swab samples. Autofluorescence from the swab matrix significantly interfered with fluorescence polarization measurements. However, quantitative detection of H5-AIV was achieved by suppressing the matrix effect through 80-fold dilution. The limit of detection was 186.4 μg mL-1. The assay can be completed within 20 minutes without complex sample preparation, making it suitable for rapid on-site applications, including field surveillance of AIV in wild birds and poultry. Furthermore, by designing appropriate tracers, the method can be extended to other AIV subtypes.