Abstract: Objective: This study aimed to optimize the extraction process of polysaccharides from Xinjiang gray jujube, characterize their monosaccharide composition, and explore its potential biological activity by integrating computational simulation and in vitro experiments. Methods: Polysaccharides were extracted from Xinjiang gray jujube using ultrasonic-assisted hot water extraction, and the extraction parameters were optimized by single-factor experiments combined with response surface methodology. After purification, monosaccharide composition was determined by ion chromatography. Potential protein targets of the major monosaccharide components were predicted using a deep learning model, followed by construction of a protein–protein interaction (PPI) network and gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and disease ontology (DO) enrichment analyses. Molecular docking was performed to assess the binding characteristics between core targets and monosaccharides. In vitro antioxidant activity was evaluated using DPPH and ABTS⁺ radical scavenging assays, and the effects of the polysaccharides on GES-1 cell viability were further assessed. Results: The optimal extraction conditions were as follows: Ultrasonic power 600 W, liquid-to-solid ratio 1:40 g/mL; ultrasonic time 11 min, water bath temperature 72 ℃, and water bath time 196 min. Under these conditions, the polysaccharide yield reached 4.856%. The polysaccharides were mainly composed of glucose (88.59%), with smaller amounts of arabinose (1.58%), galacturonic acid (2.89%), galactose (2.40%), and rhamnose (4.54%), indicating that they are acidic heteropolysaccharides containing multiple neutral sugars and galacturonic acid. Target prediction and enrichment analyses suggested that their potential effects were closely associated with glucose metabolism, the insulin signaling pathway, and glycosylation-related processes, implying potential roles in modulating glucose metabolism and ameliorating insulin resistance. In vitro experiments demonstrated concentration-dependent scavenging activities against both DPPH and ABTS⁺ radicals. Within the concentration range of 62.5~250 μg/mL, the polysaccharides showed no obvious cytotoxicity toward GES-1 cells and significantly promoted cell viability. Conclusion: Polysaccharides from Xinjiang gray jujube can be obtained at a relatively high yield after process optimization and exhibit a defined monosaccharide profile, antioxidant activity, and good cytocompatibility. They may also possess potential bioactivity related to glucose metabolism regulation. These findings provide a basis for the further development and utilization of Xinjiang gray jujube polysaccharides.