Lactobacillus acidophilus LA-G80, isolated from the human intestinal flora, has probiotic functions. In this study, the medium composition and culture conditions for this strain were optimized. The results obtained showed that the optimal medium consisted of glucose 41.5 g/L, maltose 20 g/L, soybean oligosaccharide 10 g/L, beef extract powder 18.64 g/L, tryptone 10 g/L, yeast peptone 15 g/L, Tween-80 1 mL/L, K2HPO4·7H2O 2 g/L, NaAc·3H2O 5 g/L, triammonium citrate 2 g/L, MgSO4·7H2O 0.25 g/L, MnSO4·4H2O 0.05 g/L, aspartic acid 0.16 g/L, alanine 0.16 g/L, serine 0.16 g/L, peanut protein powder 5 425 g/L, and soybean cake powder 7 425.48 g/L. The optimal culture conditions were as follows: inoculum size 2%, initial pH 6.0, constant pH 5.5, and constant temperature 37 ℃. Experiments carried out in a 5 L fermenter gave a viable cell count of 42.2 × 108 CFU/mL, which was about 10 times higher than that of observed with MRS medium.