Steady-state and time-resolved fluorescence spectroscopy were employed in the discrimination of cervical cancer patients from healthy subjects using urine samples. Fluorescence emission at 390 and 440 nm was considered to monitor the fluorescence of indoxyl sulfate and neopterin. Significant spectral differences were observed between healthy and cancer subjects. Different ratio parameters were calculated from the spectral intensity at 280- and 350-nm excitation and were subjected to stepwise linear discriminant analysis. In total, 84.0% of samples were correctly classified at 280 nm and 96.4% were correctly classified at 350 nm. The fluorescence decay kinetics of urine samples at 390-nm emission was best described by bi- exponential fits, whereas the decay characteristics at 440 nm of urine samples was best explained by bi-exponential fits and, in some cases, by tri-exponential fits. However, the decay kinetics of both indoxyl sulfate and neopterin standards was well described by bi-exponential decays. Based on the fluorescence emission characteristics and statistical analysis, the fluorophores indoxyl sulfate, neopterin, and riboflavin may be considered as potential biomarkers for cervical cancer diagnosis.