(1) Background: There is a risk of Aflatoxin B1(AFB1) pollution in yellow rice wine[1],threatening human life and health, the risk is a major focus of government attention and social concern.The current detection technology cannot meet the needs of large-scale testing due to the disadvantages of high false positive, cumbersome operating procedures or high cost.Therefore, it is urgent to study and establish a high-sensitivity and high-fast detection method for AFB1 to provide technical support for yellow rice wine quality supervision and risk assessment.; (2) Methods: In this study, three-factor and three-level orthogonal experiments were carried out for oscillation time, reaction temperature and tomography time to determine the best reaction conditions for pretreatment.AFB1 detection was carried out by aflatoxin time-resolved fluorescence immunochromatography technology,Through the ratio of the T signal value of the detection line to the C signal value of the quality control line and the natural logarithmic value of the standard solution concentration[2], a rapid detection technology of time-resolution fluorescence immunochromatography is established.The technology was methodically assessed, the actual yellow rice wine samples were tested, and the results were compared with the HPLC method; (3) Results: The results show that the optimal reaction conditions for pretreatment are: the sample oscillation time is 20min, the sample reaction temperature is 37°C, and the sample chromatography time is 6min. In the actual sample detection of yellow rice wine, The time-resolved fluorescence immunochromatography detection technology established in this study detects AFB1. The detection limit is 0.3 μg·kg-1,The linear range is 0.8-12.0μg·kg-1,The linear equation of the corresponding standard curve y=-0.1913x+0.5206 (R2=0.9948),The standard deviation is 0.029.The results of the in-batch accuracy and precision of this method show that the addition and recovery rate is 77.9% - 105.7%,The coefficient of variation is in 4.5% - 10.3%; Inter-batch accuracy and precision test results show that the addition recovery rate is75.9% - 85.8%,the coefficient of variation is in 8.1% - 13.9%,explain that the detection technology has good accuracy and precision.Compared with HPLC, the relative error is less than 10%,It shows that aflatoxin time-resolution fluorescence immunochromatography technology is in good agreement with the test results of national standard methods; (4) Conclusions:he rapid detection technology of time-resolution fluorescence immunochromatography of AFB1 in yellow rice wine is easy to operate with fast detection speed, high sensitivity, good repeatability and stability, and has broad application prospects.