Conformation-induced formation of a series of unique Raman marker bands in cancer cell DNA, upon dehydration, have been probed for the first time with the use of surface enhanced Raman spectroscopy (SERS). These bands are capable of distinguishing cancer cell DNA from healthy cell DNA. For this simple and label-free DNA detection approach, we used conventional spherical silver nanoparticles, at a high concentration, without any aggregating agents, which gave highly reproducible SERS spectra of DNA separated from various human cells irrespective of their highly complex compositions and sequences. The observed phenomenon is attributed to the change in the chemical environment due to the presence of nucleobase lesions in cancer cell DNA and subsequent variation in the nearby electronic cloud during the dehydration-driven conformational changes. Detailed analysis of the SERS spectra gave important insight about the lesion-induced structural modifications upon dehydration in the cancer cell DNA. These results have widespread implications in cancer diagnostics, where SERS provides vital information about the DNA modifications in the cancer cells.