Objective To establish a method to determine chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to investigate the application of urine glycosaminoglycans detection in the diagnosis and follow-up of mucopolysaccharidosis (MPS) patients. Methods Twenty urine samples from MPS patients and 37 urine samples from normal children were collected, and glycosaminoglycans levels in these samples were determined. Urine samples were added and dried under nitrogen. Then the samples were derivatized with methanol hydrochloride and dried under nitrogen. After reconstituted, the glycosaminoglycans were analyzed by LC-MS/MS. The linearity, limit of quantification, precision, spiked recoveries, and matrix effect were evaluated. Results The linearity of CS, DS and HS by LC-MS/MS was>0.99. The limits of quantification for CS, DS and HS were 0.5 mg/L, 1 mg/L and 1 mg/L, respectively; the intra-day imprecision ranged from 1.9% to 10.4%, and the inter-day imprecision ranged from 2.6% to 9.8%; and the recovery ranged from 85.6% to 110.4%, the relative matrix effect ranged from 84.9% to 115.5%. CS, DS and HS in urine samples of normal children were normally distributed, then the upper limit of reference interval were preliminarily determined by mean+1.64 SD, which were 16.5 mg/mmol, 1.8 mg/mmol and 1.4 mg/mmol, respectively. Urine GAGs of MPS patients were analyzed, the LC-MS/MS method was effective in detecting MPSⅠ, MPSⅡ, MPSⅢ and MPSⅥ patients. Urine samples of MPSⅠ or MPSⅡ patients who had hematopoietic stem cell transplantation showed reduced DS and HS concentration. Conclusion LC-MS/MS has good performance in detecting urinary mucopolysaccharides and is expected to be used for accurate diagnosis and treatment follow-up monitoring of MPS patients.