We demonstrate a nanoporous membrane device integrated with an on-chip microfluidic platform for the electrokinetic separation of biomolecules. This platform offers a thin (500 nm) film of anodized aluminum oxide directly fabricated and suspended onto a silicon substrate, assembled into a compact microfluidic device. We successfully showed the electrokinetic transport of ssDNA through the nano-porous membrane under various conditions. Size exclusive biomolecular separation driven by electric field was verified with the complex of thrombin and thrombin aptamer. This architecture enables an on-chip device for binary separation and size exclusive filtration targeted to various applications such as molecular detection and purification. (C) 2013 Elsevier B. V. All rights reserved.