Onosma hookeri Clarke. var. longiforum Duthie (OHC-LD), one of the traditional Tibetan medicine, has been found many functions, including removing heat to cool blood, nourishing lung and inhibiting bacteria. In order to study the polysaccharides in OHC-LD water extract, the optimal extraction progress of polysaccharides of the roots of OHC-LD by response surface method designed with three-factor three-level Box-Behnken method and the antioxidant capacity and immune activity of the crude polysaccharide were studied in this investigation. Under the best conditions, the extraction yield of polysaccharide was 3.19±0.09% (n = 3). After purification, the crude polysaccharide was obtained with polysaccharide contents of 42.57%, which demonstrated stronger DPPH scavenging activity than BHT at low concentrations (<625 µg/mL), and comparable ABTS radical scavenging activity as BHT at high concentrations (≥1250 µg/mL). Additionally, it also exhibited a certain cell proliferation activity and an enhancement of the phagocytic ability of RAW264.7 cells. This study revealed that the crude polysaccharide from the roots of OHC-LD might be exploited as a natural antioxidant and immune enhance agent in the future in both medical and food industry.