Abstract: To optimize the polysaccharides extraction process from Hemerocallis citrina Baroni and investigate the hypoglycemic activity of polysaccharides in vitro, the plasma-activated water (PAW) was employed in conjunction with ultrasound treatment to improve the polysaccharides extraction rate. The optimum extraction rate was determined by using a single-factor experiment and response surface design. Then, the polysaccharides were purified using Sephadex G-100 dextran gel chromatographic columnto obtain the pure polysaccharides from Hemerocallis citrina Baroni (HCP). The structure of HCP was identified and its hypoglycemic activity was evaluated by the approval α-glucosidase and α-amylase inhibition experiments. The results indicated that the optimal process parameters were liquid-solid ratio of 23 mL/g, extraction time of ultrasound 60 min, PAW preparation time of 90 s, in which the maximum yields of Hemerocallis citrina Baroni polysaccharides was 36.33%±0.04%. The total sugar content, protein content, and uronic acid content of HCP were 88.07%±2.25%, 2.72%±0.04%, and 7.27%±0.44%, respectively. HCP was an acidic heteropolysaccharide with α-pyranose configuration, consisting of fucose, rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid. The molecular weight of HCP was 8.056 kDa. The SEM showed that HCP was sheet-like with a rough surface. Additionally, the HCP exhibited great hypoglycemic activity in vitro and the concentration-dependent. When the concentration was 1 g/mL, the inhibition ability of α-glucosidase and α-amylase reached 83.02% and 78.65% respectively. This experiment indicated that HCP had the potential to become functional raw materials for hypoglycemic preparations.