Objective To develop a rapid, highly sensitive quantitative method for detecting P24 antigen based on near-infrared fluorescent microsphere immunochromatography.Methods First, we prepared a lateral flow assay test strip, and labeled the detection antibody using a fluorescent microsphere. Second, we optimized the antibody labeling conditions. Third, we optimized the detection conditions. Fourth, we created a working curve. Fifth, we conducted a methodological assessment of the established fluorescent microsphere immunochromatography method. Sixty-six clinical samples were tested, and we compared the established fluorescent microsphere immunochromatography with the quantitative ELISA method. Results According to the working curve, the detection limit of the method is 3.4 pg/mL, and the detection range is 3.4 pg/mL to 10 ng/mL. The average intra-assay recovery was 99.6％, and the Coefficient of Variation (CV) was 5.4％–8.6％; the average inter-assay recovery was 97.3％, and the CV was 8.5％–11％. The detection rate of fluorescent microsphere immunochromatography was higher than ELISA method, and had a good correlation with ELISA. Conclusion The P24 antigen quantitative detection method based on near-infrared fluorescent microsphere immunochromatography has the advantages of rapid detection, high sensitivity, and wide detection range; thus, it is suitable for early clinical diagnosis and continuous monitoring of AIDS.