Considering global economic and environmental benefits, green renewable biofuels such as ethanol and butanol are considered sustainable alternatives to fossil fuels. Thus, developing a co-culture strategy for ethanol and butanol production by Saccharomyces cerevisiae and Clostridium beijerinckii has emerged as a promising approach for biofuel production from lignocellulosic biomass. This study developed a co-culture of S. cerevisiae and C. beijerinckii for ethanol and butanol production from non-detoxified corn stover hydrolysate. By firstly inoculating 3% S. cerevisiae and then 7% C. beijerinckii with 8-10 h time intervals, the optimized co-culture process gave 24.0 g/L ABE (20.8 g/L ethanol and 2.4 g/L butanol), obtaining ABE yield and productivity of 0.421 g/g and 0.55 g/L/h. The demonstrated co-culture strategy made full use of hexose and pentose in hydrolysate and contributed to total yield and efficiency compared to conventional ethanol or ABE fermentation, indicating its great potential for developing economically feasible and sustainable bioalcohols production.