Techniques that manipulate DNA, a biomolecule with electrical properties, are in demand in various medical fields. This study fabricated a nanochannel with a conductive/semi-conductive interface using focused ion beams (FIBs) and introduced a nanochip technology to freely align, attach, and detach lambda DNAs in the interface via electrophoresis. Two-step fabrication process of nanochannels was quantitatively characterized according to the different conditions of the FIB dose (1~30 nC/μm2) and current (1~500 pA). For electrophoresis test, four different nanofluidic channels with depths of 200 nm and lengths of 0.5, 1.0, 1.5, and 2.0 μm were processed at the center of the rectangular channel (10 μm × 10 μm). Different voltages (1~30 V) were applied for 15 min to attach the DNAs. As the voltage increased, more lambda DNAs attached to the nanochannel interface. Furthermore, an inverse voltage (-30 V) was applied to the lambda DNAs attached to the interface for 15 min to confirm that DNAs could be successfully detached. The results showed that this method could produce a highly promising nanochip technology to align and manipulate DNAs in the desired direction according to a conductive/semi-conductive nano-sized interface, which is applicable in various biomedical fields.