Strain Lactobacillus fermentum MC3 isolated from fermented bamboo shoots was used in this study. This isolate had a high exopolysaccharide (EPS) production capability. It was inoculated in the medium with a supplement of various concentrations of carbohydrate source (2, 3, 4, 5, and 6% (w/v)). The mixtures were then cultured under different conditions of initial cell density, temperatures, pH, and incubation time to identify the optimum param-eters for the EPS biosynthesis of this strain. The EPS yield was measured using the phenol-sulfuric acid method. The results showed that adding glucose, lactose, and sucrose to the cul-ture medium significantly increased the EPS production with a maximum amount of 4% of sugars. The yield was the highest for glucose at 178.207 mg/L, and the obtained figures for lactose and sucrose were 148.614 mg/L and 152.272 mg/L, respectively. The results indicated that the EPS production by L. fermentum MC3 reached the maximum values at 200.728 mg/L in the medium supplemented with 4% glucose at 40 °C, pH 6.0, and initial cell density of 106 CFU/mL for 48 h cultivation. By methylation and gas-chromatography mass spectrometry (GC-MS), it was found that the exopolysaccharide is composed of D-mannose, D-glucose, and D-galactose at the molar ratio of 1:0.74:0.09