The methodological development of nucleic acids detection is a rapidly growing research field. Here, we report a powerful method to detect nucleic acids by an integration of surface-enhanced Raman scattering and exonuclease III-assisted probe amplification. With a unique signal-on strategy, we have demonstrated that the target DNA of MnSOD gene in concentrations as low as 1 aM can reproducibly be detected, which offers a detection limit several orders of magnitude better than the previous reports in the literature. The new biosensor exhibits an excellent specificity in differentiating DNA sequences with a single-base mismatch. As a robust, flexible, and ultrasensitive approach, it promises important applications in clinical diagnostics and DNA identification where only a very limited amount of the biological sample is available.