In this paper, the optimal extraction conditions of crude polysaccharide from Hippophae rhamnoides L. were gained by response surface methodology (RSM) according to the principle of Box-Behnken design (BBD), and three purified fractions (PBP-1, PBP-2 and PBP-3) were isolated by multiple column chromatography. PBP-1 mainly contained arabinose, xylose, mannose, glucose and galactose in a molar ratio of 1.18: 1: 2.20: 32.17: 1.45. PBP-2 mainly contained xylose, mannose, glucose and galactose in a molar ratio of 1: 0.28: 1.02: 0.2. PBP-3 mainly contained xylose, glucose and galactose in a molar ratio of 1: 2.15: 0.28, and the average molecular weights were 26.5, 5.6 and 4.9 kDa, respectively. Furthermore, the CPBP and PBP-3 exhibited the higher effective scavenging activities against DPPH, ABTS and reducing power, and the CPBP indicated the highest protective effect against H2O2-induced oxidative injury in LO2 cells. These findings could provide useful information for the utilization and development of the resources of Hippophae rhamnoides L.