Abstract In order to study the composition, structure and physicochemical properties of Tremella fuciformis polysaccharide (TFPS), four new polysaccharide fractions (TFPS1, TFPS2, TFPS3 and TFPS4) were successfully isolated from the hot-water extracted crude polysaccharides by DEAE-52 cellulose column. Their structures were analyzed by FT-IR, SEM, HPGPC, monosaccharide composition, Congo red and I2-KI experiments. TFPS1 was a heteropolysaccharide with a molecular weight of 742 Da for the major polysaccharide fragment. TFPS2, TFPS3 and TFPS4 were single component homogeneous polysaccharides with 350774 Da, 622675 Da and 624724 Da, respectively. Glucose was the major monosaccharide of TFPS1 and TFPS2, while Mannose was the major monosaccharide of TFPS3 and TFPS4. TFPS1 and TFPS2 displayed a stable triple helix structure at aqueous solution, while TFPS3 and TFPS4 presented longer side chains and more branched chains. TFPS1, TFPS2, TFPS3 and TFPS4 all possessed in vitro antioxidant activities. Moreover, the four polysaccharide fractions all demonstrated significant cholesterol binding ability, as well as obvious sodium taurocholate binding ability. The results of this study may provide some guidance on the medicinal value of TFPS.