Beta-glucan is a polysaccharide derived from mushrooms’ cell wall mainly used for increase the immune system. Besides, beta-glucan is also used to reduce the blood glucose level which can be done by doing beta-glucosidase inhibition test. This research was aimed to isolate and characterize crude beta-glucan from Tremellafuciformis,as well as assess its bioactivity. Hexane and ethyl acetate were used to remove impurities prior to hot water extraction to get the isolated beta-glucan, resulting in 2.580.33%. C-NMR, H-NMR, and FT-IR were used to confirm the characteristic of crude beta-glucan. C-H and CH2 groups were detected within δH 3.5-4.2 ppm, shown by H-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 CNMR 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 beta-glucan contained glucose, fucose, mannose, melibiose, and Dgalactose. Antidiabetic activity was measured by performing alpha-glucosidase inhibitory evaluation. Crude beta-glucan was found to have a weak activity on alpha-glucosidase since it has lower inhibitory activity than quercetin. The crude beta- glucan started to show its inhibitory activity at 50 ppm by resulting yellow transparent color with the highest activity of 41.612.25%, i.e. lower than quercetin as the positive control (71.410.25%).