Urolithiasis is a common disease with high recurrence rate. According to the record, the prevalence rate of urolithiasis is about 4–15% in Asia, Europe and America while the recurrence rate of urolithiasis is more than 50% after the treatment. The research done by the University of Chicago indicates that the presence of crystals in urine is an important factor of stone formation. Previous studies also conclude that the compound of crystals and stones are highly correlated. Therefore, it is important to analyze urine crystals accurately to prevent potential stone formation. Our Raman spectroscopic urine crystal detection system features the specially designed microfluidic chip with a chamber, the patented technology of crystal collection using Fe3O4 nanoclusters and the 785-nm excitation wavelength automatic Raman microscope we constructed. This system can instantly extract crystals from urine, and the composition of crystals are determined accurately by Raman spectroscopy. It will be a much more powerful and more efficient tool on urine crystal analysis. The clinical significance study of urine crystals on urolithiasis management mainly focuses on the relationship between morphology including auto-fluorescence of urine crystals and urolithiasis. The urine samples and renal calculi from urolithiasis patients were collected from Taipei Veterans General Hospital and Taipei City Hospital and all the samples were analysized with our Raman spectroscopic urine crystal detection system. Statistically, the pre-surgery urine samples of urolithiasis patients tend to have more atypical crystals in shape, composition and auto-fluorescence incidence than non-patients’. Our data indicates that the pattern of urine crystals plays a significant role in clinical urolithiasis management.