Monitoring of the epidemic situation is imperative to control the risk of infection with avian influenza H5 and H7 subtype viruses. A microneutralization (MN) assay was employed to detect hemagglutinin (HA) subtype-specific antibodies. However, the conventional MN assay raises biosafety concerns and is labor-intensive and time-consuming. Therefore, a safer and more convenient assay that can be applied in a high-throughput format is warranted. In this study, PB2 knockout (PB2-KO) influenza viruses of H5 and H7 subtypes expressing different colored fluorescent proteins were generated using a reverse genetics system and applied to a novel MN assay for the detection of specific antibodies. The detection sensitivity of our PB2-KO virus-based MN assay was evaluated by observing fluorescent proteins under a fluorescence microscope and measuring fluorescence intensities using a plate reader. In addition, the PB2-KO virus-based MN assay was used for the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Expression of the reporter fluorescent protein from H5 and H7 PB2-KO viruses was restricted toin PB2 protein-expressing cells. The MN titer as determined using fluorescence microscopy and plate reader revealed that the detection sensitivity of our PB2-KO virus-based MN assay was comparable to that of the conventional MN assay. Moreover, H5 and H7 PB2-KO viruses could be usedapplied forto the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Our study demonstrates a safe and convenient assay for the detection of H5 and H7 subtype-specific antibodies.