Abstract: The polysaccharides were extracted from the dried fruits of Rosa roxburghii Tratt fruit (RPs), and the structure and bioactivity of RPs were investigated, which provided the basis for the development and utilization of RPs resources. The extraction was conducted by using an ultrasound-assisted enzymatic method combined with macroporous resin decolorization and trichloroacetic acid (TCA) deproteinization. The extracted RPs were structurally characterized by using high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectrophotometer (UV-Vis), scanning electron microscope (SEM), and atomic force microscope (AFM). The antioxidant activity, cholesterol esterase inhibitory activity, and anticancer activity of RPs were also determined. The results showed that the amounts of carbohydrates of RPs were 52.31%±3.56%, the galacturonic acid content was 45.53%±2.75%, and the protein content was 2.0%±0.07%. RPs were mainly composed of galacturonic acid, galactose, rhamnose, arabinose and a small amount of fructose, glucose, xylose and glucuronic acid, and the molar mass percentage was 43.65%, 18.67%, 15.39%, 16.34%, 0.92%, 1.79%, 1.02% and 2.22%, respectively. RPs showed scavenging rates of 84.4%, 99.5%, and 10.4% for DPPH, ABTS⁺, and hydroxyl radicals, respectively, when the concentration of RPs was 2.0 mg/mL. The concentrations for 50% of maximal effect (EC₅₀) were 1.93, 0.53, and 7.83 mg/mL, respectively. Furthermore, the cholesterol esterase inhibition rate was 48.6% when the concentration of RPs was 1.0 mg/mL. The CCK-8 assay indicated that RPs inhibited the growth of oral squamous carcinoma Cal27 cells without affecting normal fibroblast L929 cells. In conclusion, the RPs extracted by this method possessed diverse biological activities and had potential applications in functional foods or pharmaceutical products.