Exopolysaccharides of L. actobacillus plantarum (LAB EPS) have become a research focus due to their health-promoting properties. The purpose of this study was to develop a highly efficient and simple separation technology for LAB EPS. A simple aqueous two-phase system (ATPS) based on polyethylene glycol (PEG)/NaH2PO4 was first applied to isolate exopolysaccharides from L. actobacillus plantarum 1.0665. The distribution behavior of LAB EPS derived from the ATPS was studied using a single-factor test combined with the response surface method. Effective separation of LAB EPS was achieved by ATPS consisting of 22.5% PEG 600 (w/w) and 17.0% NaH2PO4 (w/w) at pH = 6.0, with a K of 5.21 ± 0.05 and a Y of 71.57 ± 0.39%. The structural characteristics of the obtained LAB EPS were subsequently examined. The LAB EPS had a molecular weight of 95276 Da, consisting of mannose, glucose and galactose at molar ratios of 1.00, 0.65 and 0.20, respectively. An ordered helical conformation maintained by hydrogen bonding interactions was identified in the LAB EPS molecules. The biological activity assay revealed significant antioxidant activity, anti-gout activity and hypoglycemic activity of LAB EPS. In addition, LAB EPS was kinetically characterized as a competitive inhibitor of α-amylase and α-glucosidase.