An aptamer affinity column (AAC) was constructed for alternariol (AOH) extraction and purification in traditional Chinese medicines (TCMs). AAC was constructed through loading aptamer functionalized Sepharose that conjugated through NHS-activated Sepharose and amino group modified aptamer into an extraction column. AOH could be captured efficiently when the sample solution flowed through AAC because of the affinity between aptamer and AOH, and the absorbed AOH was then eluted with the elution solvent for further high-performance liquid chromatography (HPLC) analysis. The conditions that influence the extraction efficiency, including acetonitrile percentage, sample loading volume, washing, and elution solvents, were carefully studied. Under the best conditions, AAC exhibited a maximum adsorption amount of AOH at 5.0 μg, also good specificity and excellent reusability (could be reused at least 10 times) were obtained. Finally, AAC was successfully applied in AOH extraction in six kinds of real TCM samples with satisfactory recoveries ranging from 90.2 to 105.7%, with RSD less than 7.5%. Moreover, the constructed AAC was compared with a commercial immunoaffinity column, and it exhibited significantly better recoveries. The proposed AAC, with its excellent properties, demonstrated potential for target purification and detection in TCMs analysis through the specific binding between aptamer and its related target.