Aflatoxins are a class of highly toxic chemical contaminants occurring in food. Consumption of aflatoxin-contaminated food can lead to harmful effects on human health. A rapid and reliable analysis of aflatoxins in food is crucial. In this study, we generated a broad–specific monoclonal antibody (MAb) 3 C10 against aflatoxin B1 (AFB1). The MAb 3 C10 binds specifically to AFB1 and AFM1 and has a IC50 of 0.13 μg L−1 for AFB1 and 0.16 μg L−1 for AFM1. Furthermore, the MAb showed high cross-reactivity to AFB2, AFG1, and AFG2. To enable simultaneous AFB1 and AFM1 detection in different food matrices, an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on MAb 3 C10 has been developed and optimized. In addition, the extraction methods of different food matrices (peanut, corn, soybean, wheat flour, rice, soy sauce, vinegar, wine, raw milk, pure milk, skimmed milk, and yogurt) were established. The average recovery ranged from 73 to 121 %, with relative standard deviation values less than 15 %. The limit of detection was 0.52 + 0.36 μg kg−1 (mean + 3SD) for AFB1 in eight agricultural products and 0.031 + 0.015 μg kg−1 (mean + 3SD) for AFM1 in four dairy products. The sensitivity of icELISA was below the limit set by the European Commission for aflatoxin detection in different food matrices and similar to LC–MS/MS method. We demonstrate a rapid, simple, and reliable method for simultaneous screening of AFB1 and AFM1 in different food matrices.