Isolating a particular strand of DNA from a double stranded DNA duplex is an important step in aptamer generation as well as many other biotechnology applications. Here we describe a microfluidic, flow-through, dialysis device for isolating single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). The device consists of two channels fabricated in polydimethylsiloxane (PDMS) separated by a track etched polycarbonate membrane (800 nm pore size). To isolate ssDNA, dual-biotin labelled dsDNA was immobilized onto streptavidin-coated polystyrene beads. Alkaline treatment was used to denature dsDNA, releasing the non-biotinylated ssDNA. In the flow-through dialysis device the liberated ssDNA was able to cross the membrane and was collected in an outlet channel. The complementary sequence bound to the bead was unable to cross the membrane and was directed to a waste channel. The effect of NaOH concentration and flow rate on purity and yield were compared. >95% ssDNA purity was achieved at 25 mM NaOH. However, lower flow rates were necessary to achieve ssDNA yields approaching the 50% theoretical maximum of the concurrent-flow device. Under optimized conditions the microfluidic isolation achieved even higher purity ssDNA than analogous manual procedures.