Abstract: β-glucan is a polysaccharide derived from mushrooms’ cell wall mainly used for increase the immune system and it is also used to reduce the blood glucose level. This research was aimed to isolate and characterize crude β-glucan from Tremella fuciformis, as well as assess its bioactivity by doing α-glucosidase inhibition test. Hexane and ethyl acetate were used to remove impurities prior to hot water extraction to get the isolated β-glucan, resulting in 2.58±0.33%. 13C-NMR, 1H-NMR and FT-IR were used to confirm the characteristic of crude β-glucan. C-H and CH2 groups were detected within δH 3.5-4.2 ppm, shown by 1H-NMR. Meanwhile anomeric proton that indicated β-glycosidic linkage was shown at 4.8-5.2 ppm which was also confirmed by FT-IR in 1028 cm-1 region. The most abundant spectra shown by 13C-NMR in chemical shift δC 71.58; 71.87; 72.90; 73.08; 73.47; 76.53; followed by δC 60.41; 60.95; indicated CH and CH2. Anomeric carbon that indicated glycosidic carbon was shown at 93.13-102.01 ppm. FT-IR showed the OH group at 3500-3000 cm-1, followed by 1250-1122 cm-1 for -C-O and 1500-1400 cm-1 for C-H absorption. In addition, methyl group which belong to fucose was shown by δH at 1.07 and 1.21 ppm which also conformed by the result of GC-MS showed the crude β-glucan contained glucose, fucose, mannose, melibiose and D-galactose. Antidiabetic activity was measured by performing α-glucosidase inhibitory evaluation. Isolated crude β-glucan was found to have a weak activity on α-glucosidase since at 50 ppm, it has lower inhibitory activity than quercetin.