To study the structure of β-glucans, we developed a separation method and molecular library of β-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a β-glucan of Laminaria digitata . They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the β-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer β-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the β-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of β-glucans.