A novel structure of exopolysaccharide from the Lactic Acid Bacteria (LAB) Lacticaseibacillus rhamnosus ACS5, isolated from home-made Turkish cheese, is described. After lyophilization, the L-EPS-ACS5 was characterized in production and functional activities in vitro, including antioxidant and antidiabetic activities. The physicochemical characterizations of the L-EPS-ACS5 were determined through molecular weight, UV, FTIR, SEM, TGA, HPLC, NMR, methylation, and GC–MS analysis. Strong antioxidant activities of L-EPS-ACS5 were confirmed from the results obtained in the hydroxyl radical, DPPH, ABTS, FRAP, superoxide anion radical, total antioxidant activity, and DNA damage protective effect, and also the L-EPS-ACS5 exhibited high antidiabetic activity (60 %). This study isolated L-EPS-ACS5 from a home-made cheese L. rhamnosus strain, demonstrating its novel and enhanced functionalities compared to existing strains. This opens exciting avenues for its development in the fields of biomedicine and pharmaceuticals.