We have developed an aptamer affinity column (AAC) for the purification and enrichment of trace aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in genuine agro-products through the covalent conjugation of amino modified aptamer and NHS-activated Sepharose. The coupling and working conditions found to be suitable for this AFB-AAC were examined in regard to coupling time (2 min), loading volume (30 mL), and the methanol concentration (< 10%) used in the loading step. The performance of AFB-AAC was then further evaluated in terms of capacity (329.1 ± 13.7 ng for AFB1 and 162.5 ± 8.9 ng for AFB2), selectivity (excellent), reusability (twenty-three times for AFB1 and twelve times for AFB2), and repeatability (92.7% ± 2.9% for AFB1 and 71.5% ± 3.4% for AFB2). Furthermore, the AAC clean-up combined with HPLC-FLD demonstrated excellent linearity over a wide range, good sensitivity with an LOD of 50 pg mL-1 for AFB1 and 15 pg mL-1 for AFB2, and acceptable recovery with different spiking levels in different matrices. Finally, the AAC was successfully applied to analyte AFB1 and AFB2 in four types of agro-products as well as a maize flour reference material, and the results were found to be in accordance with those of commercial IACs. This study provides a reference for the analysis of other trace analytes by merely changing the corresponding aptamer and represents a strong contender for immune affinity columns. Graphical abstract An aptamer affinity column for purification and enrichment of aflatoxin B1 and aflatoxin B2 in agro-products with the aid of HPLC-FLD and a post-column photochemical derivatization reactor.