Sol-gel-based mesopores allow the entry of target small molecules retained in their cavity and aptamers to bind to target molecules. Herein, sol-gel-based materials are applied to screen-selective aptamers for small molecules, such as pesticides. To enhance the efficiency of aptamer screening using a sol-gel, it is necessary to increase the binding surface. In this study, we applied the sol-gel to an anodized aluminum oxide (AAO) membrane, and the morphological features were observed via electron microscopy after spin coating. The binding and elution processes were conducted and confirmed by fluorescence microscopy and polymerase chain reaction. The sol-gel coating on the AAO membrane formed a hollow nanocolumn structure. A diazinon-binding aptamer was bound to the diazinon-containing sol-gel-coated AAO membrane, and the bound aptamer was effectively retrieved from the sol-gel matrix by thermal elution. As a proof of concept, a sol-gel-coated AAO disc was mounted on the edge of a pipette tip, and the feasibility of the prepared platform for the systematic evolution of ligands by exponential enrichment (SELEX) of the aptamer binding was also confirmed. The proposed approach will be applied to an automated SELEX cycle using an automated dispenser, such as a pipetting robot, in the near future.