Aflatoxin B1 (AFB1) is a toxic compound naturally produced by the genera Aspergillus. Distillers' grains can be used as animal feed since they have high content of crude protein and other nutrients. However, they are easily contaminated by mycotoxins, and currently there are no rapid detection methods for AFB1 in distillers' grains. In this study, a lateral flow immunoassay (LFIA) based on red fluorescent microsphere (FM), is developed for quantitative detection of AFB1 in distillers" grains. The whole test can be completed within 15 min, with the cut-off value being 25.0 μg/kg, and the quantitative limit of detection (qLOD) being 3.4 μg/kg. This method represents satisfactory recoveries of 95.2-113.0%, and the coefficients of variation (CVs) are less than 7.0%. Furthermore, this technique is successfully used to analyze AFB1 in real samples, and the results indicates good consistency with that of high-performance liquid chromatography (HPLC). The correlation coefficient is found to be greater than 0.99. The proposed test strip facilitates on-site, cost-effective, and sensitive monitoring of AFB1 in distillers" grains. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.