Human norovirus is the leading cause of non-bacterial gastroenteritis worldwide in all age groups. In this study, a rapid, high-sensitivity and quantitative detection method for VP1 protein of norovirus GII was developed based on time-resolved fluorescence microsphere immunochromatography. The optimal labeling amount and coated antibody concentration of norovirus monoclonal antibody were 10 μg and 1.5 mg mL-1, respectively. The best detection time was 15 min after sample addition. The detection of NoV GII VP1 protein had a good linear relationship in the range of 2.5 ng mL-1-320 ng mL-1 (Y = 0.6784X - 1.1443, R2 = 0.9935), and the lowest limit of detection was 0.61 ng mL-1. There was no cross reaction with rotavirus type A and enteric adenovirus type 40 lysate. In 88 clinical samples, the positive coincidence rate was 97.06%, the negative coincidence rate was 96.27%, and the total coincidence rate was 96.60%. The area under the subject operating characteristic curve was 0.9670 and the 95% confidence interval was (0.9289,1.000). Therefore, this study established a rapid, sensitive and wide detection range of GII human norovirus detection method, which can provide an auxiliary diagnostic method for early clinical diagnosis of norovirus and population screening.