Abstract OBJECTIVE To establish a detection method for fingerprints and content determination of free sugar and hydrolyzed monosaccharide of intracellular glycopeptides in Coriolus versicolor. METHODS PMP derivatization after ultrasonic extraction with water as solvent was determined for free sugars, and PMP derivatization after acid hydrolysis was determined for hydrolyzed monosaccharides. The free sugars and hydrolyzed monosaccharides of raw materials and intermediates of intracellular glycopeptides from different manufacturers were analyzed by HPLC qualitatively and quantitatively. The fingerprints were analyzed by chemometrics analysis, and the differences between intracellular glycopeptides produced by different manufacturers were discussed. RESULTS Both two methods were validated by methodology. The samples from different manufacturers were clustered separately, and three free sugars and two hydrolyzed monosaccharides contributed greatly to the quality difference of intracellular glycopeptides of Coriolus versicolor. CONCLUSION The established analytical method can be effectively used for the determination of free sugars and hydrolyzed monosaccharides in the intracellular glycopeptide of Coriolus versicolor and the study of fingerprints. The adopted chemometrics research method provides guidance for the quality control of the intracellular glycopeptide of Coriolus versicolor.