Abstract: Objective To establish a micro-plaque reduction assay to detect human serum level of neutralizing antibody against avian influenza A (H7N9) virus. Methods MDCK cells were infected with H7N9 virus, plaques were measured to determine the titer of the virus. The optimal infectious dose of the H7N9 virus was calculated, human sera in serial dilutions were added to the virus at optimal infectious dose, the number of plaques reduced was measured, and the neutralizing antibody titer to H7N9 virus was determined. Results Plaque reduction method was used to detect anti H7N9 neutralizing antibody level, which had no cross reaction with seasonal influenza, avian influenza H5N1pdm09, H9N2 subtype virus antibodies. Micro-plaque reduction method showed consistent results with micro-neutralization method. Conclusion Micro-plaque reduction method had good sensitivity and specificity in detecting human serum level of neutralizing antibody against avian influenza A (H7N9) virus.