Urine is one of the diagnostically important bio fluids, as it has different metabolites in it, where many of them are native fluorophores. Native fluorescence characteristics of human urine samples were studied using excitation–emission matrices (EEMs) over a range of excitation and emission wavelengths, and emission spectra at 405 nm excitation, to discriminate patients with cancer from the normal subjects. The fluorescence spectra of urine samples of cancer patients exhibit considerable spectral differences in both EEMs and emission spectra with respect to normal subjects. Different ratios were calculated using the fluorescence intensity values of the emission spectra and they were used as input variables for a multiple linear discriminant analysis across different groups. The discriminant analysis classifies 94.7% of the original grouped cases and 94.1% of the cross-validated grouped cases correctly. Based on the fluorescence emission characteristics of urine and statistical analysis, it may be concluded that the fluorophores nicotinamide adenine dinucleotide (NADH) and flavins may be considered as metabolomic markers of cancer.