In order to meet the needs of rapid quantitative detection of fumonisin B1 (FB1) in grains, this paper takes fumonisin B1 as the research object, and creates a rapid quantitative detection card of FB1 on the basis of time-resolved fluorescent nanospheres, with no need to adjust the PH of samples before detection. The detection performance of the detection card in grains (rice, corn, wheat) was analyzed and compared with enzyme-linked immunosorbent assay kit and high performance liquid chromatography. The LOD for different samples was in the range of 35.37~37.17 μg/kg, and the LOQ was 117.9~123.9 μg/kg, which meant good sensitivity; the linear range was 250~6 000 μg/kg, while the correlation coefficient R2 was in the range of 0.997 2~0.999 5; the spiking recoveries at 6 spiked levels in different samples were in the range of 83.38%~114.66%, with the coefficient of variation (CV) less than 15%, which meant good accuracy and repeatability. When the national standard liquid chromatography and FB1 fluorescence quantitative rapid detection card simultaneously detects different samples contaminated by FB1, the coincidence of the detection results was in the ranger of 92.53%~106.26%, with the CV less than 10.37%. The cross-reaction rate of other five common mycotoxins was less than 5%. Meanwhile, the difference between the spiked concentration and their average detected concentration was extremely significant, indicating good specificity. Hence, the FB1 fluorescence quantitative detection card prepared in this study could meet the needs of on-site rapid quantitative detection of fumonisin B1 in grains.