A water-soluble polysaccharide, named GPN, with molecular mass 38.7 kDa was isolated from Glycyrrhiza glabra with hot water extraction, ethanol precipitation, and purified by column chromatography. Monosaccharide composition analysis confirmed the presence of predominant glucose (98.03%) and trace amount of mannose, arabinose, and galactose. Methylation and GC-MS analysis revealed that the main glycosidic bonds in GPN comprised 1,4-linked Glcp, T-linked Glcp, 1,4,6-linked Glcp, and 1,6-linked Glcp. Based on these results and 1D/2D NMR spectroscopy, GPN has a linear backbone of 1,4-linked alpha-D-Glcp and 1,6-linked alpha-D-Glcp which substituted at C-4 of glucose. The side chain probably composed from 1,4-linked to main side alpha-D-Glcp and terminal 1-linked beta-D-Glcp to the C-3 of D residue. Congo red assay confirmed the existence of triple helix structure. Moreover, SEM and XRD analysis revealed that the GPN had irregular fibrous, filaments like surface; and both crystalline and amorphous structure. GPN also displayed favorable thermal stability. Moreover, G.glabra polysaccharide showed good antioxidant activity.