Abstract: To establish the purification process of polyphenols from Shitougan (Citrus reticulata Blanco cv. Manau Gan) peel and explore their functional activities, macroporous resins were initially screened based on their adsorption and desorption performance, and the purification conditions were subsequently optimized. In addition, in vitro assays were conducted to evaluate the antibacterial, hypoglycemic, anti-inflammatory, and antioxidant activities of the purified fractions. The results revealed that the D101 resin exhibited the highest enrichment efficiency for the polyphenols in Shitougan peel. The optimized purification parameters were as follows: When the column volume was 15 mL, the mass concentration of the sample solution was 20 mg/mL, the flow rate was 1.0 mL/min, the sample volume was 30 mL, the elution flow rate was 1.5 mL/min, and the volume of the eluent was 40 mL. After purification, the polyphenol content rose from 32.91 mg/g (in the crude extract) to a range of 67.01~193.6 mg/g, and the antibacterial, hypoglycemic, anti-inflammatory, and antioxidant activities were significantly enhanced (P<0.05). Nevertheless, disparities were observed among the fractions obtained through gradient eluate with varying ethanol concentrations and the overall eluate with 70% ethanol. In the gradient elution process, the diameters of the inhibition zones of the 20% ethanol elution fraction against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Bacillus subtilis were 19.29±0.32, 21.27±0.57, 22.34±0.27, and 17.56±0.43 mm, respectively. The IC₅₀ value of the inhibition rate of NO production was 0.57 mg/mL, and the IC₅₀ value of the scavenging rate of ABTS⁺ free radicals was 0.052 mg/mL, both of which were significantly superior to those of other purified products and the crude extract (P<0.05). The IC₅₀ value of the 40% ethanol elution fraction for the inhibition rate of α-glucosidase was determined to be 16.51 μg/mL, and the IC₅₀ value of the 70% ethanol eluate for the scavenging rate of DPPH free radicals was found to be 0.307 mg/mL. Both values were significantly superior to those of other purified products and crude extracts (P<0.05). The purification process parameters proposed in this study can be applied to the extraction and production of polyphenols from the peel of Shitougan, offering a theoretical foundation and technical reference for the development and high-value comprehensive utilization of the peel of Shitougan.