A simple and sensitive method for DNA detection using gold nanoparticle (AuNP) two-probe detection system (AuNP-TP) was developed. Preliminary experiment was carried out by optimizing slide types, blocking agents and hybridization times. Fluorescent-labeled probes were used along with AuNP-labeled probes to confirm specific binding event between target DNA and probes. The sensitivities between AuNP single-probe (AuNP-SP) and AuNP-TP systems using sandwich-typed assay were compared. The AuNP-TP on epoxide-coated (EP) slides increased sensitivity 1000-fold at the detection limit of 100 fM when compared to the AuNP-SP. This result indicates that the assay sensitivity was simply enhanced by simultaneous adding two AuNP labeled probes which selectively recognize different regions of the target DNA. The concept of AuNP-TP could potentially be applied to a macroarray format to detect multiple DNA targets simultaneously; thereby making the assay becomes more affordable and more sensitive.