To enhance the production of bacteriocin DF01, produced by Lactobacillus brevis DF01, cultivation conditions and medium composition were optimized by using response surface methodology (RSM). The selected 5 factors based on MRS medium were glucose, yeast extract, $MgSO_4$, temperature, and initial pH. Fractional factorial design (FFD) was effective in searching for the main factors. By a $2^{5-1}$ FFD, glucose, yeast extract, and initial pH were found to be significant factors and had positive effects on bacteriocin production. The effects of the 3 main factors on bacteriocin production were further investigated by a central composite design (CCD). RSM revealed that the maximum bacteriocin production was achieved at yeast extract concentration of 14.56 g/L, glucose concentration of 28.95 g/L, and initial pH of 6.8. After RSM, the titer of bacteriocin was increased by 4-fold.