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BACKGROUND Mycotoxins are secondary metabolites produced by Fungi, which have serious effects on humans and animals. In this study, we selected the monodispersed polystyrene fluorescent microspheres with good luminescence performance and strong stability as markers to conjugate with four mycotoxins antibodies for preparing fluorescent probes. We have developed a fluorescent microsphere based immunochromatographic assay (FMICA) to detect sensitively and quickly zearalenone (ZEN), aflatoxin B1 (AFB1 ), fumonisin B1 (FB1 ), and ochratoxin A (OTA) in cereal. RESULTS Under optimal experimental conditions, the procedure of this method can be completed within 10 min. The limit of detection (LOD) of FMICA for ZEN, AFB1 , FB1 , and OTA is 0.072, 0.093, 0.32, and 0.19 μg·L-1 , respectively. And FMICA has good specificity and no cross-reactivity with other mycotoxins. Four mycotoxins in naturally contaminated cereal samples (corn, rice, and oat) are detected by this method, and the results are highly consistent with that of UPLC-MS /MS. CONCLUSION The developed FMICA has good accuracy, high sensitivity, simplicity, convenience, rapidity, and low cost, and it could be employed for sensitive and quantitative detecting of mycotoxins in cereal on-site. This article is protected by copyright. All rights reserved.