Triple helical polysaccharides (t-polysaccharides) are easily gelated in water, resulting in difficult fractionation, leading to the complex and time-consuming chain conformational characterization. Moreover, the fractionation is not always successful due to the coexistence of individual chains and aggregates. In this work, we developed a facile and reliable method to rapidly and accurately characterize the chain conformation of t-polysaccharide without fractionation needed in traditional conformation characterization. A triple helical beta-1,3-glucan (t-beta-1,3-glucan), extracted from the fruiting bodies of Lentinus edodes, was identified to consist of a beta-1,3-glucan with two beta-1,6-D-glucopyranoside branchings for every five beta-1,3-glucopyranoside linear linkages by one- and two-dimensional NMR and GC-MS analysis. The chain conformations of the t-beta-glucan in aqueous solution and in DMSO were successfully characterized by a combination of size exclusion chromatography (SEC), multiangle static light scattering, a differential refractometer, and a capillary viscosity detector (triple-detector SEC). The results revealed that the predominate species of the t-beta-glucan in a 0.15 M NaCl aqueous solution existed as a triple helical conformation with high chain stiffness, and a few aggregates (4%) coexisted here. The Mark-Houwink and (S-2)(1/2) versus M-w equations of individual triple helical chains and aggregates were obtained simultaneously, and the results confirmed again the coexistence of two kinds of chain conformations. The fractal dimension indicated that the aggregate in the aqueous solution was a kind of reversible microgel with a 3D network structure. Furthermore, the chain morphology of the t-beta-glucan in aqueous solution was observed directly by transmission electron microscopy and atomic force microscopy to support the worm-like chain for the individuals and 3D network for the aggregates. The triple-detector SEC technology was facile and reliable for the system with two fractions of different chain conformation, and the test time required was only 1/30 of what the traditional method needed.