Stokes shift spectroscopy (S3) is an emerging approach toward cancer detection. The goal of this paper is to evaluate the diagnostic potential of the S3 technique for the detection and characterization of normal and cancerous prostate tissues. Pairs of cancerous and normal prostate tissue samples were taken from each of eight patients. Stokes shift spectra were measured by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval $\mathrm{\Delta }\lambda =20\mathrm{nm}$ between them. The salient features of this technique are the highly resolved emission peaks and significant spectral differences between the normal and cancerous prostate tissues, as observed in the wavelength region of 250 to 600 nm. The Stokes shift spectra of cancerous and normal prostate tissues revealed distinct peaks around 300, 345, 440, and 510 nm, which are attributed to tryptophan, collagen, NADH, and flavin, respectively. To quantify the spectral differences between the normal and cancerous prostate tissues, two spectral ratios were computed. The findings revealed that both ratio parameters $R_1=I_{297}/I_{345}$ and $R_2=I_{307}/I_{345}$ were excellent diagnostic ratio parameters giving 100% specificity and 100% sensitivity for distinguishing cancerous tissue from the normal tissue. Our results demonstrate that S3 is a sensitive and specific technique for detecting cancerous prostate tissue.