Abstract: Objective: To optimize the extraction process of polysaccharides from Plumeria rubra L. cv. Acutifolia, and make acomment about antioxidant, antibacterial and anti-tumor activities of Plumeria polysaccharide. Methods: The orthogonal experiment was designed by single factor experiment and the optimal extraction conditions were determined. The antioxidant activity was evaluated by measuring its scavenging ability to DPPH, ABTS, and OH free radical assays. The minimum inhibitory concentration of Plumeria polysaccharide against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae was measured by the micro broth dilution method to evaluate the antibacterial activity of Plumeria polysaccharide. In addition, CCK-8 assay was used to evaluate the inhibitory effect of Plumeria polysaccharide on human melanoma cells (A375), mouse melanoma cells (B16), human colorectal cancer epithelial cells (DLD-1) and human breast cancer cells (MCF-7). Results: The optimum conditions of Plumeria polysaccharide were as follows:Ultrasonic power was 264 W, solid-liquid ratio was 1:50 (g/mL), extraction time was 40 min, extraction temperature was 35 ℃. Under these conditions, the yield of Plumeria polysaccharide was 14.01%±0.22%. The half-inhibitory concentrations of Plumeria polysaccharide in scavenging DPPH, ABTS and OH free radicals were 0.1934, 0.2315 and 2.469 mg/mL, respectively. The minimum inhibitory concentrations of Plumeria polysaccharide against Escherichia coli and Staphylococcus aureus was 0.391 and 0.195 mg/mL, respectively, and the minimum inhibitory concentrations against Pseudomonas aerugino, Klebsiella pneumoniae and Acinetobacter baumannii were all 1.562 mg/mL. The results of cell viability experiments showed that within a certain concentration range, except for DLD-1 cells, Plumeria polysaccharide had a certain inhibitory effect on the viability of A357, B16 and MCF-7 cells and was dose-dependent with the concentration, with IC₅₀ of 101.3, 285.6 and 423.1 μg/mL. Conclusion: This article optimizes the extraction process of Plumeria polysaccharides for the first time. A series of experiments prove that Plumeria polysaccharide has good antioxidant, antibacterial and anti-tumor activities in vitro. This research provides a certaindata theoretical support for the further development and utilization of Plumeria.