Microfluidic chip with nano-scale structures has shown great potential, but the fabrication and cost issues restrict its application. Here, we propose a conceptually new “DNA nanolithography in microfluidic chip” by using sub-10 nm three-dimensional DNA structures (TDNs) as frameworks with a pendant aptamer at the top vertex (ApTDN-Chip). The nano-scale framework ensures the aptamer with a highly ordered upright orientation, avoiding the undesired orientation or crowding effects caused by conventional microfluidic interface fabrication processes. Compared with a monovalent aptamer modified chip, the capture efficiency of ApTDN-Chip was enhanced nearly 60% due to the highly precise dimension and rigid framework of TDNs. In addition, the confined tetrahedral nanostructure scaffolds make DNase I more accessible to aptamer with up to 83% release efficiency and 91% cell viability, which is fully compatible with downstream molecular analysis. Overall, “DNA nanolithography in microfluidic chip” provides a novel perspective to engineer nano-scaffolds to achieve a more ordered nano-topography of microfluidic chip.