Abstract: Rhamnogalacturonan II (RG-II) is one of the structural domains of pectin whose structure is highly conserved among species. The main chain of RG-II consists of approximately nine galacturonic acids linked by α-1,4 glycosidic bonds, with six well-defined side chains replacing them (A−F). The structures of the disaccharide side chains C and D and the monosaccharide side chains E and F in RG-II from different sources remain essentially the same. In contrast, the oligosaccharide side chains A and B showed slight variability. Structural characterization of RG-II can be achieved by molecular weight, monosaccharide composition, and mass spectrometry. The polysaccharides containing RG-II structural domains in traditional Chinese medicines (TCMs) have high medicinal value. Isolation of RG-II can be achieved using endo-polygalacturonase (Endo-PG) and Penicillium oxalicum. A substantial number of RG-II standards can be rapidly prepared from red wine for the development of new quantitative methods to realize the quality control of active polysaccharides from traditional Chinese medicines and to promote the research process of polysaccharides from traditional Chinese medicines.