Exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a linear dextran that is built by glucose units via α-1,6 glycosidic bond. The primary objective of this study was to investigate the yield of XG5 EPS and its application in set yogurt. In laboratory scale, the culture conditions of XG5 EPS production were optimized using the L9 (3 3 ) orthogonal test. Here, the optimized yield of XG5 EPS was 26.02 g/L under the conditions of 100 g/L sucrose, initial pH 7.0, 25°C incubation, and 100 rpm for 36 h in a shaking flask. Based on the optimized parameters of laboratory scale, a pilot fed-batch fermentation was performed in a 50-L bioreactor with an adjusted agitation speed of 20 rpm. The XG5 EPS yield reached 40.07 g/L in fedbatch fermentation, which was 54% higher than that achieved in laboratory scale. In addition, XG5 EPS was added into set yogurt to investigate its effect on the stability of set yogurt. Our data demonstrated that the XG5 EPS improved the water-holding capacity, texture profile, and viscosity of set yogurt during cold storage compared with the controls. In particular, addition of 0.5% XG5 EPS increased the structure of 3-dimensional network of set yogurt, which eventually improved the physical stability of the set yogurt. Overall, this study provided new insights for exploring the pilot scale production and application of dextran.