| Summary: | Aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) is a highly teratogenic and carcinogenic secondary metabolite produced by <i>Aspergillus</i>. It is commonly detected in agricultural products such as cereals, peanuts, corn, and feed. Grains have a complex composition. These complex components severely interfere with the effective extraction and separation of AFB<sub>1</sub>, and also cause problems such as matrix interference and instrument damage, thus posing a great challenge in the accurate analysis of AFB<sub>1</sub>. In this study, an aptamer affinity column for AFB<sub>1</sub> analysis (AFB<sub>1</sub>-AAC) was prepared for the enrichment and purification of AFB<sub>1</sub> from grain samples. AFB<sub>1</sub>-AAC with an AFB<sub>1</sub>-specific aptamer as the recognition element exhibited high affinity and specificity for AFB<sub>1</sub>. Grain samples were enriched and purified by AFB<sub>1</sub>-AAC, and subsequently analyzed by high performance liquid chromatography with post-column photochemical derivatization-fluorescence detection (HPLC-PCD-FLD). The average recoveries of AFB<sub>1</sub> ranged from 88.7% to 99.1%, with relative standard deviations (RSDs) of 1.4–5.6% (<i>n</i> = 3) at the spiked levels of 5.0–20.0 μg kg<sup>−1</sup>. The limit of detection (LOD) for AFB<sub>1</sub> (0.02 μg kg<sup>−1</sup>) was much below the maximum residue limits (MRLs) for AFB<sub>1</sub>. This novel method can be applied to the determination of AFB<sub>1</sub> residues in peanut, corn, and rice.
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