Chirality is one of the fundamental biochemical properties in a living system, and a lot of biological and physiological processes are greatly influenced by the chirality of molecules. Inspired by this phenomenon, we study the covalent assembly of DNA on chiral molecule modified surfaces and further discuss the hybridization of DNA on chiral surfaces with nucleic acids. Take methylene blue (MB) modified DNA as a model molecule, we show that the peak current of the L-NIBC (NIBC, N-isobutyryl-L(D)-cysteine) modified gold surface (L-surface) is larger than the D-surface because of a stronger interaction between short-chain DNA and the L-surface; however, the D-surface has a higher hybridization efficiency than the L-surface. Moreover, we apply this result to actual application by choosing an electrochemical DNA (E-DNA) sensor as a potential platform. Furthermore, we further amplify the difference of hybridization efficiency using the supersandwich assay. More importantly, our findings are successfully employed to program the sensitivity and limit of detection.