Distillers' grains (DGS), the main waste by-products of Chinese liquor industry, were used as substrate for surfactin production. Bacillus amyloliquefaciens MT45 could grow with DGS as sole carbon source to produce 1.04 g/l surfactin. However, low amylase activity of MT45 limited sugar supply and the subsequent surfactin production. Therefore, MT45 was co-cultured with Bacillus strains that exhibited remarkable hydrolases activities. Surfactin yield increased by 50% when MT45 was co-cultured with B. amyloliquefaciens X82 that showed no product inhibition effect and did not develop extracellular matrix. The inoculation ratio of X82 greatly influenced the sugar supply, cellular growth, and surfactin production of the co-culture fermentation. Maximum surfactin titration (3.4 g/l) was obtained when MT45 and X82 were co-cultured with inoculation ratio at 1: 0.5, using 200 g/l DGS. This work highlights the feasibility of using industrial waste DGS as promising feedstocks to produce value-added surfactin by co-culture fermentation. (C) 2017 Elsevier Ltd. All rights reserved.