Abstract: Abstract: Fuel ethanol production from lignocellulosic biomass hydrolysates by microbial fermentation was of great economic and environmental significance. Distillers grains, which are composed of unhydrolyzed and unfermented polymeric sugars, are the co-products of white spirit industries, their high polysaccharides portion of cellulose and hemicellulose make them attractive feedstock for conversion to fuel ethanol. However, biomass recalcitrance that limit the availability of polysaccharides for biological conversion by enzymatic digestibility necessitates a appropriate pretreatment, which is in favor of hydrolytic enzyme penetration to enhance enzymatic digestibility. The aim of this study is to increase accessibility of cellulolytic enzymes (xylanase and cellulase) to the feedstock and evaluate the effects of different pretreatment methods on enzymatic saccharification of distillers grains biomass. Four pretreatment processes including ultrasound-assisted acid pretreatment (UAAP), soaking in aqueous ammonia pretreatment (SAAP), alkaline hydrogen peroxide pretreatment (AHPP) and bisulfite pretreatment (BP) were employed in this study to determine how each method affected the digestibility of distillers grains during enzymatic hydrolysis. The effective mechanisms of four different pretreatment methods on the chemical composition and structural characteristics were studied and analyzed with the goal of removing lignin and reserving of holocelluloses (cellulose and hemicellulose). Then the effects of four different pretreatment methods on degradation efficiency of cellulose and hemicellulose to produce sugars were evaluated with the index of the enzymatic hydrolysis yield. SEM and XRD were applied to characterize structural changes associated with improved cellulolytic enzyme digestibility. The results showed that the retention rate of cellulose and hemicellulose by BP pretreatment was higher than that of other three pretreatment methods, which were 84.59% and 84.87%, respectively. Four pretreatment methods effectively enhanced enzymatic hydrolysis efficiency by disrupting lingocellulosic structure and increasing accessibility of cellulolytic enzymes to distillers grains over un-pretreated feedstock. Compared with the un-pretreated distillers grains, the enzymatic hydrolysis yield of holocellulose were increased by 49.12% (UAAP), 55.48% (PASS), 92.79% (AHPP) and 99.15% (BP), respectively. Compared with other three pretreatment approaches, BP pretreatment showed the greatest improvement on enzymatic hydrolysis of the distillers grains. The maximum conversion of holocellulose was 32.88%, which were 1 times of the control (un-pretreated 16.51%). SEM data showed that the structural modifications of 4 pretreatments were pronounced in comparison to that of feedstock, which presented a rigid and compact morphology. Remarkably, most of the large irregular pores with different sizes and shapes were formed and the lignocellulosic structure was also severely disrupted as a result of the removal of lignin, leading to the increasing surface exposure and porosity. Furthermore, 4 pretreatments disrupted the internal structure of the holocelluloses, increased the reaction surface area and the porosity of the distillers grains. The results based on the analysis of XRD suggested that these pretreatments disrupted the crystal structure of cellulose and increased the available surface area, which made the cellulose better accessible for enzymatic hydrolysis. The changes of physical structure and chemical composition declare that the enzymatic digestibility is related with the removal of lignin, the retention or disorder structure of holocelluloses and the crystallinity of cellulose. Therefore bisulfite pretreatment (BP) is effective and appropriate for the pretreatment of distillers grains to increase the enzyme saccharification yield.