The Stokes shift spectroscopy (S3) offers a simpler and better way to recognize spectral fingerprints of fluorophores in complex mixtures. The efficiency of S3 for cancer detection in human tissue was investigated systematically. The alterations of Stokes shift spectra (S3) between cancerous and normal tissues are due to the changes of key fluorophores, e.g., tryptophan and collagen, and can be highlighted using optimized wavelength shift interval. To our knowledge, this is the first time to explicitly disclose how and why S3 is superior in comparison with other conventional spectroscopic techniques.