An antibody-modified Teslin paper was prepared in combination with direct mass spectrometry for the rapid and sensitive analysis of trace aflatoxin B1 (AFB1) in grain and its related products. Antibodies were covalently immobilized to Teslin surface by glutaraldehyde method, and an immunoaffinity paper substrate (IAP) was obtained. IAP can not only be used as a carrier for enrichment of analytes, but also coupled with the MS inlet for desorption/ionization detection. Compared to antibody-free Teslin, IAP could have excellent selectivity for the enrichment of AFB1. It took less than 1 min for detection. Among the matrices (wheat flour, peanut oil and corn), the proposed method showed satisfactory selectivity and linearity. The LOD and LOQ were 0.40–0.80 μg kg−1 and 1.20 μg kg−1, respectively. In conclusion, the IAP can provide a new powerful tool for rapid and sensitive analysis of mycotoxins.