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Systematic evolution of ligands by exponential enrichment (SELEX) is a method that is generally used for developing aptamers, which have arisen the promising alternatives for antibodies. However, conventional SELEX methods have limitations, such as a limited selection of target molecules, time-consuming and complex fabrication processes, and labor-intensive processes, which result in low selection yields. Here, we used (i) graphene oxide (GO)-coated magnetic nanoparticles in the selection process for separation and label-free detection and (ii) a multilayered microfluidic device manufactured using a three-dimensionally printed mold that is equipped with automated control valves to achieve precise fluid flows. The developed on-chip aptamer selection device and GO-coated magnetic nanoparticles were used to screen aptamer candidates for adenosine in eight cycles of the selection process within approximately 2 h for each cycle. Based on results from isothermal titration calorimetry, an aptamer with a dissociation constant of 18.6 ± 1.5 μM was selected. Therefore, the on-chip platform based on GO-coated magnetic nanoparticles provides a novel label-free screening technology for biosensors and micro/nanobiotechnology for achieving high-quality aptamers. [ABSTRACT FROM AUTHOR]