This study aims to furnish diagnostic evidence and insights for fever of unknown origin (FUO) patients through the analysis of urine proteomics. Urine samples from FUO patients were subjected to one-to-many comparative analysis utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS) to identify differential proteins and associated biological pathways. One–to–many analysis means a comparative analysis of one sample to many controls. Our findings revealed biological pathways pertinent to fever, including LXR/RXR activation, FXR/RXR activation, and acute phase response signaling, thereby demonstrating that urine analysis can effectively differentiate between disease states and health. Furthermore, the variability observed among individual samples underscores the importance of employing a one-to-many analytical approach. The integration of urine proteomics with one-to-many analysis offers valuable insights for the investigation of FUO and may also be applicable in the study of other undiagnosed diseases.