Abstract: Cordyceps militaris polysaccharides from the fermentation broth were prepared via chitosan flocculation and water extraction and alcohol precipitation methods to obtain the flocculated polysaccharide (XDT) and alcohol precipitation polysaccharide (CDT) respectively. The extraction yields, component content, average particle sizes and solubility of XDT and CDT were comparatively investigated. The microstructures, functional groups and spatial conformations were characterized by scanning electron microscopy, Fourier infrared spectroscopy and Congo red assay. The antioxidant activity was evaluated by scavenging ability of DPPH, superoxide anion and hydroxyl radical. The hypoglycemic activity was estimated by α-glucosidase inhibitory ability, and the antitumor cell activity was analyzed by inhibition of HepG2 cell proliferation. The results showed a yield of 3.58% for XDT, 1.91 times that of CDT. CDT and XDT exhibited different microstructures under electron microscopy, and similar absorption patterns under infrared spectrum, while XDT contained more triple helix structures than CDT. XDT displayed smaller average particle sizes but a faster dissolution rate than CDT. In vitro antioxidant tests showed that XDT exhibited significantly higher scavenging ability of DPPH, superoxide anion and hydroxyl radical than CDT at the same concentration. There was no significant difference between the XDT and CDT in the inhibition of α-glucosidase at low concentration, but XDT showed significantly higher inhibition than CDT as the increase of concentration. XDT was found to be a stronger inhibitor of HepG2 cells proliferation than CDT at the same treatment concentration. In summary, XDT showed higher yields, smaller average particle sizes, faster solubility, and higher antioxidant, hypoglycemic, and antitumor activity in vitro.