Surface-enhanced Raman spectroscopy (SERS) has been successfully used for the label-free detection of single-stranded oligonucleotides. However, the detection of complex DNA secondary structures remains a challenge. Structural features of diverse DNA G-quadruplexes were investigated via a novel SERS method. As a result, a series of highly reproducible and sensitive SERS signatures featuring the structures of G-quadruplexes were obtained. For the first time, we reported remarkably enhanced SERS bands corresponding to purine ring breathing vibrations. Moreover, we observed that by measuring the intensity of the bands corresponding to intramolecular hydrogen bonds, we could quantitatively assess the stability of the G-quadruplexes. Because no labels on DNA strands were present as the experiments were carried out in the solution, the fingerprint peaks reflect the native, internal structure of the G-quadruplexes accurately. The method here detailed provides new insights into the promising applications of diverse DNA structural studies.