It is generally believed that the self-folding of single-stranded DNA depends on the hydrophobic effect of its internal bases, but the folding of a single-stranded DNA in a solution was not disordered and would be affected by the stacking effect of adjacent bases. In this work, we developed a new method to explore the stacking between adjacent bases using Surface-Enhanced Raman Spectroscopy (SERS) for the first time. Acidic titanium ions were introduced into silver nanoparticles as an aggregating agent (Ag@ITNPs), and obtained a symmetrical spectrum by normalizing the peak to deoxyribose at 955 cm-1. Based on the influence of adjacent base stacking on the spectrum, we first identified the point mutation sites accurately by SERS. Also, the base content and the DNA frameshift mutations in ssDNA were precisely analyzed. This new method has a simple experimental process and can accurately capture the changes in the base ring breathing peak intensity caused by different adjacent bases, and thus will provide potential application value in the field of gene diagnosis.