A DNA configuration switch is designed to fabricate a reversible and regenerable Raman-active substrate. The substrate is composed of a Au film and a hairpin-shaped DNA strand (hot-spot-generation probes, HSGPs) labeled with dye-functionalized silver nanoparticles (AgNPs). Another ssDNA that recognizes a specific trigger is used as an antenna. The HSGPs are immobilized on the Au film to draw the dye-functionalized AgNPs close to the Au surface and create an intense electromagnetic field. Hybridization of HSGP with the two arm segments of the antenna forms a triplex-stem structure to separate the dye-functionalized AgNPs from the Au surface, quenching the Raman signal. Interaction with its trigger releases the antenna from the triplex-stem structure, and the hairpin structure of the HSGP is restored, creating an effective "off-on" Raman signal switch. Nucleic acid sequences associated with the HIV-1 US long terminal repeat sequences and ATP are used as the triggers. The substrate shows excellent reversibility, reproducibility, and controllability of surface-enhanced Raman scattering (SERS) effects, which are significant requirements for practical SEAS sensor applications.