The Tremella fuciformis Berk polysaccharides (TP) have significant cytokine-stimulating activity and low oral bioavailability owing to their large molecular volume. Identifying and studying the smallest active structure of TP can potentially be beneficial for further utilization and research, therefore, TP was hydrolyzed and fractionated to obtain its (1→3)-mannan backbone fragments. The fragments were further isolated by ion-exchange resin, ultrafiltration, gel, and HPLC chromatography, and three oligosaccharide fractions, named TL4-1, TL4-2, TL4-3, were obtained. According to the determination of their physicochemical properties, they were composed of Man, and after methylation analysis they were mainly characterized as (1→3)-mannan oligosaccharides with a straight chain. TL4-1 contained di-, trisaccharides, TL4-2 included tetra-, penta-saccharides, and TL4-3 was made up of hex-, hepta-, octa-saccharides, according to LC-ESI/MS analysis. Cytokine stimulation experiments showed that the degree of polymerization (DP) should be more than six to maintain the activity of mannosan. Therefore, the smallest active unit of oligo-mannose was determined. PRACTICAL APPLICATIONS: This paper reports the physical and chemical parameters, structure and biological potential of oligosaccharides from T. fuciformis Berk, a common edible fungus. Tremella has been used as an improving immunity drug in China with good effect. Oligosaccharides are more easily digested and utilized by human body, and maintain good activity. These results can increase people's interest in the product, and thus have a positive impact on the oligosaccharides of Tremella as health food.