Abstract: The extraction condition of polysaccharide from red pulp Hylocereus undatus stem ( PPL) by subcritical water was optimized using the single factor and orthogonal experiment. Determination of antioxidant activity and composition of polysaccharide were studied to determine the biological activity of red pulp Hylocereus undatus stem and evaluate its vitro antioxidant activity, so that we can provide theoretical basis for the rational development and application of red pulp Hylocereus undatus stem polysaccharide.The results showed that the optimal extraction conditions were that the extraction temperature was140 ℃, the time was 25 min and the solid-liquid ratio was 1∶ 50, and the extraction rate of PPL was up to 38.07% ± 0.15%.The study on antioxidant revealed that the PPL had certain antioxidant activity and the maximum scavenging rate on hydroxyl radical and DPPH radical were 40.09% and 95.15%, respectively.Ultraviolet and infrared spectrum analysis showed that PPL had the typical polysaccharide IR absorption.