Abstract: The time-resolved fluorescence immunochromatographic assay (TRFIA) was established by competitive inhibition for determination of deoxynivalenol (DON).Nanometer microspheres with Eu³⁺ chelate were chosen as fluorescent probes using for tagging DON monoclonal antibody.This quantitative method was studied in the application of mycotoxins detection to determine the sensitivity, precision and accuracy.The results showed that for each 100 μL fluorescence microspheres combined with pure single antibody 50 μg, the best film concentration was 1.0 mg/mL, the optimal measurement conditions were room temperature (25 ±2)℃, 10 min, loading volume 100 μL, buffer solution of PBS (0.01 mol/L, pH7.2), the detection limit was 0.25 ng/mL, the linearity range was 0.5~25.0 ng/mL, and the average recovery rate was 91.4%~109.3% respectively when the concentration of standard addition was 200, 500, 1000 and 2000 μg/kg.The content of DON was detected in different samples such as corn and wheat by TRFIA and immune affinity-high performance liquid chromatography (IAC-HPLC) method simultaneously.The correlation coefficient (r) was 0.9754 comparing these two methods.Therefore, TRFIA was characterized by high sensitivity, fast speed and quantitative accuracy, which could be applied to the quick determination of DON in cereals and derived products.