In this study, a novel polysaccharide was isolated from Artemisia ordosica by water-extraction-ethanol-precipitation method. The optimal extraction conditions of Artemisia ordosica polysaccharide (AOP) were determined by single factor investigation and response surface methodology optimization, and were shown as follows: a liquid–solid ratio of 15.4 : 1 mL g⁻¹, extraction time of 4.3 h, extraction temperature of 60 °C. Under the optimal conditions, the extraction yield and the sugar content of the AOP were 5.56% and 52.65%. Gel permeation chromatography coupled to multi-angle laser light scattering, a refractive index detection system and ion-exchange chromatography were used to determine the characterization of AOP. These results indicated that AOP, with a molecular weight of 2.1 kDa (62.6%) and 1.5 kDa (37.4%), had narrow polydispersity and rod conformations, and was composed of arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid with molar ratio of 6.87 : 10.67 : 54.13 : 2.49 : 18.37 : 4.83 : 2.64 : 2.64. In addition, AOP exerted antioxidant ability in vitro and in vivo (rats). Moreover, AOP significantly modulated the composition of cecal microbiota population. Therefore, AOP is expected to be a functional ingredient for health improvement through improving antioxidant ability and modulating gut health.