Small structural differences bring great difficulties on carbohydrates identification, especially in terms of their quantification. Herein, a novel ESI-MS/MS based strategy was established to discriminate and relatively quantified protonated PMP-disaccharides with different composition and glycosidic bond. Interestingly, protonated PMP labeled-disaccharides provided abundant fragment ions arising from cross-ring cleavage and glycosidic bond cleavage, which could afford diagnostic fragment patterns for isomers differentiation in combination of statistical analysis. It was worth to note that the relative intensity ratios (RIR) of three ion pairs could completely discriminate 16 disaccharides, and subsequently used to relatively quantified isomers in a binary mixture. Ultimately, this method was applied for the discrimination of yellow rice wine, and then the relative content of maltose and isomaltose were confirmed as well. In general, this method was easy to operation and effective for rapid differentiation and quantification of isomeric disaccharides in complex matrices.