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Immunoassay has the advantages of high sensitivity, high specificity, and simple operation, and has been widely used in the detection of mycotoxins. For several years, time-resolved fluorescence immunochromatography (TRFIA) paper-based sensors have attracted much attention as a simple and low-cost field detection technology. However, a traditional TRFIA paper-based sensor is based on antibody labeling, which cannot easily meet the current detection requirements. A second antibody labeling method was used to amplify the fluorescence signal and improve the detection sensitivity. Polystyrene fluorescent microspheres were combined with sheep anti-mouse IgG to prepare fluorescent probes (Eu-IgGs). After the probe fully reacted with the antibody (Eu-IgGs-Abs) in the sample cell, it was deployed on the paper-based sensor using chromatography. Eu-IgGs-Abs that were not bound to the target were captured on the T-line, while those that were bound were captured on the C-line. The paper-based sensor reflected the corresponding fluorescence intensity change. Because a single molecule of the deoxynivalenol antibody could bind to multiple Eu-IgGs, this method could amplify the fluorescence signal intensity on the unit antibody and improve the detection sensitivity. The working standard curve of the sensor was established under the optimum working conditions. It showed the lower limit of detection and higher recovery rate when it was applied to actual samples and compared with other methods. This sensor has the advantages of high sensitivity, good accuracy, and good specificity, saving the amount of antibody consumed and being suitable for rapid field detection of deoxynivalenol.