Mass spectrometry (MS) has been developed for high-sensitivity structural determination of carbohydrates. Due to the similarity of oligosaccharide structures, there is still insufficient information about the correlation between carbohydrate chemical structure and mass spectra. A series of trisaccharides containing different glycosidic linkages (1→2, 1→3, 1→4, 1→6, 2→1 and 2→6), anomeric configuration (α and β), and monosaccharide (pentose, hexose and hexuronic acid), as well as two cyclodextrins (α- and β-cyclodextrin) were systematically investigated using matrix assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI TOF/TOF MS) in positive and negative ion modes using super-DHB (2,5-dihydroxybenzoic acid) and harmine hydrochloride as matrices, respectively. Cross-ring cleavage generated by A-types ions with neutral losses of 48, 60, 78, 90, or 120 Da were observed in the negative ion MS/MS spectra, and neutral losses of 60, 90, or 120 Da were observed in the positive ion MS/MS spectra. Glycosidic cleavages were observed in both modes. The linkage isomers were unambiguously identified by those diagnostic fragment ions with their relative intensities. Furthermore, the correlation between linkage pattern and characteristic ions successfully allowed the characterization of polysaccharides with different structural features.