A carbodiimide derivative having a dithiolane part at its terminus was designed and synthesized for use to construct carbodiimide-coated self-assembly monolayers (SAMs) on a gold surface with 6-mercaptohexanol (6MH). When treated with poly(dT), poly(dA), or poly(dA)poly(dT), only poly(dT) was immobilized on the surface of the SAMs through a specific reaction of the free imino moiety of thymine (T) with the carbodiimide moiety. The carbodiimide-covered SAM treated with probe DNA was tested in hybridization with sample DNA. Its hybridization efficiency was estimated by ferrocenylnaphthalene diimide (FND), described previously and the result revealed that the carbodiimide-covered SAM electrode can immobilize a DNA probe through the thymine moiety not involved in base pairing. The resulting electrode was capable of hybridizing with the target DNA, as proven by an increased current response of FND.