<span class="paragraphSection"><div class="boxTitle">Abstract</div>Aptamers are conventionally selected via ‘Systematic Evolution of Ligands by Exponential Enrichment’ (SELEX). However, this process is laborious, time-consuming, and has a relatively low efficacy. In this study, we present a novel automated high-throughput screening platform that augments the conventional selection of DNA aptamers. To this end, the software of an optical next-generation sequencer has been modified to automatically perform fluorescence-based binding assays on the displayed DNA sequences subsequent to sequencing. Utilizing this platform, high-affinity DNA aptamers were identified for the proteins LecA, LecB, and <span style="font-style: italic;">Pseudomonas</span> Exotoxin A (PEA) of <span style="font-style: italic;">Pseudomonas aeruginosa</span> following pre-enrichment by a mere three to five SELEX rounds. Conversely, 12 rounds of conventional SELEX yielded aptamers exhibiting three-fold lower affinity for LecA and PEA, with no aptamers obtained for LecB. Furthermore, we demonstrate that the proposed method is suitable for the study of molecules ranging from small molecules to whole cells. This is evidenced by a mutation assay for a kanamycin-binding aptamer and the monitoring of <span style="font-style: italic;">Escherichia coli</span> binding to aptamers. The present study proposes a high-throughput approach to enhance SELEX, with the potential to provide greater insight into the selection process and to significantly increase efficacy, enabling the selection of aptamers within a week.</span>