This study presents a DNA detection method via hybridization of a probe oligonucleotide with a target DNA and with a substrate oligonucleotide, which leads to self-assembly of gold nanoparticles and a change in the observed current. In contrast to previously reported methods, the present method can be used to detect a target DNA without a silver enhancement step. The device can be washed with 0.3 M PBS below 50 °C for a few minutes, indicating the stringency of target DNA hybridization. Target DNA concentration of as low as 10 picomolar can be detected by this method. Importantly, identification of single-base-pair mismatch in the target DNA can be accomplished with this nano-gap DNA chip.