Abstract: Through a combined approach of single-factor experiments and response surface methodology, this research aims to optimize the ultrasound-ethanol synergistic extraction parameters for Morus nigra polysaccharides, and comprehensively characterize their bioactive potential. Employing Morus nigra as the raw material, this study systematically evaluates the individual effects of four critical parameters—temperature (40~80 ℃), ultrasonic power (120~240 W), extraction time (30~90 min), and ethanol concentration (0~50%)—on polysaccharide yield through single-factor experiments. The extraction process was optimized using Box-Behnken design response surface methodology (BBD-RSM) with 29 experimental runs (including 5 center points), establishing a predictive model for yield with ANOVA-verified parameter interactions (P<0.05). Key extraction parameters including temperature (63 ℃), ultrasonic power (180 W), time (61 min), and ethanol concentration (39%) were determined to achieve a maximum polysaccharide yield of 13.63%±0.33%. The bioactive properties of the polysaccharides were systematically evaluated through in vitro assays. Results demonstrated remarkable free radical scavenging capacity with IC₅₀ values of 0.12 mg/mL (ABTS⁺), 0.21 mg/mL (DPPH), and 2.78 mg/mL (hydroxyl radicals). Furthermore, significant enzyme inhibitory effects were observed with IC₅₀ values of 0.16 mg/mL (α-amylase), 0.24 mg/mL (α-glucosidase), and 1.64 mg/mL (lipase), suggesting potential applications in functional foods for glycemic and lipid control. Notably, the optimized polysaccharides demonstrated significant antioxidant properties coupled with potent inhibitory effects on carbohydrate-digesting and lipid-metabolizing enzymes. These findings not only establish an efficient extraction protocol but also substantiate the potential of mulberry polysaccharides as multi-functional ingredients for developing nutraceuticals targeting oxidative stress, hyperglycemia, and hyperlipidemia.