Abstract: Abstract: Bioethanol has been indispensable in future due to the consumption of fossil fuels, greenhouse effect and environmental degradation. Thus acetone-butanol-ethanol (ABE) fermentation by Clostridium species has played a vital role in production of renewable energy used to relieve the energy crisis. Renewable biomass such as corn stover as a kind of low cost raw material has replaced molasses needed for fermentation. Butanol production from corn stover hydrolysates (CSH) has obvious advantages in the field of biomass conversion. The inhibitory compounds formed during the corn stover pretreatment. Therefore detoxification of hydrolysate can be used to increase butanol production by overcoming inhibitory effects of toxic by-products such as phenols, furfural and acid. All kinds of detoxification especially the physical and chemical methods have some disadvantages because of the damage of glucose and the pollution of the environment in the downstream process. Among the different detoxification methods, enzymatic detoxification has become a high efficient and environmental-friendly biological method. In order to relieve the influence of a variety of toxic substances in the CSH on microbial growth and fermentation production, the best strategy of detoxification with laccase and formate dehydrogenase (FDH) was studied. The optimum adding content of laccase and FDH was studied to eliminate phenols and formate in hydrolysates, respectively. The results showed that ABE and butanol production from CSH dealt with 5 U/mL laccase were respectively 0.78 and 1.03 g/L by fermentation with Clostridium acetobutylicum CICC 8016, which were increased by 56% and 77.59% compared to that from non-detoxified hydrolysate, respectively. However, ABE production was lower than control group when adding laccase greater than or equal to 15 U/mL. The production from P2 medium with 0.74 g/L formate when adding 1 U/mL FDH achieved the same effect with that without adding formate medium. And ABE and butanol production from CSH dealt with 1 U/mL FDH were 0.77 and 1.11 g/L, respectively, which were increased by 57.14% and 60.87% compared with that from non-detoxified hydrolysate, respectively. Through the fermentation results with P2 and corn straw hydrolysate as medium, FDH had an obvious effect for removing formic acid in the medium. In addition, butanol production and glucose consumption were increased by 165.78% and 27.16% respectively through adsorbing treatment with 1% activated carbon. Under this condition, an efficient enzymatic composite detoxification strategy was formed assisted with activated charcoal detoxification. The enzymatic composite detoxification strategy was feasible for the CSH treated by 5 U/mL laccase, 1 U/mL FDH and 1% activated carbon after steam explosion and pretreatment of corn straw. Butanol and ABE production from composite detoxified CSH were 2.90 and 4.4 g/L respectively, which were about 5 times higher than the control group. The utilization of reducing sugar reached 98%-100%, and the yield of ABE increased by 300% compared with the control group. This research provides a reliable detoxification method for butanol fermentation with the hydrolysate of steam exploded corn straw, and the enzymatic composite detoxification strategy is more efficient and environmental-friendly than the single physical or chemical detoxification method.