Polysaccharides exhibit multiple pharmacological activities, which are closely related to their structural characteristics. Therefore, quantitative quality control of polysaccharides based on chemical properties is of importance for their applications. However, polysaccharides are mixed macromolecular compounds that are difficult to separate, and the lack of standards made direct quantification more difficult. In this study, we proposed a new quantitative method based on the released specific oligosaccharides for polysaccharides from Lentinus edodes (shiitake) and other related fungi. Specific oligosaccharides were firstly released from polysaccharides using 1,3-β-glucanase, then derivatized with 2-aminobenzamide (2-AB), which further separated by hydrophilic interaction chromatography (HILIC) and quantitatively determined by UPLC coupled with fluorescence detector (FLR). Laminaritriose was used as the universal standard for quantification of all the oligosaccharides. This method was validated according to linearity, limit of detection, limit of quantitation, precision, accuracy, repeatability and stability. In addition, the four specific oligosaccharides released from polysaccharides in L. edodes were qualitatively analyzed by extracted ion chromatogram (EIC) from UPLC-MS profiles, which were identified to be disaccharide, trisaccharide and tetrasccharide. The proposed strategy not only realized the quantitative analysis of polysaccharides by UPLC-FLR, but also could achieve the qualitative distinction of different polysaccharides.