Abstract: To study the methanogenic characteristics of lignocellulosic ethanol fermentation stillage,In this study,the performance for the two-phase AD with ethanol fermentation stillage of steam explosion pretreatment Pennisetum purpureum as the substrate was investigated and the microbial community structure of methanogenic phase was also analyzed. The results show that the main fermentation products in the acidification reactor are butyric acid and acetic acid,and the cellulose conversion rate and VS removal rate is 89% and 85.3% respectively. The acidification products are effectively degraded in the methanogenic reactor and the average methane concentration and average methane yield obtained is 69.09% and 298.5 L/Kg COD respectively. Geobacter is the major bacteria in methanogenic reactor while acetoclastic methanogens is dominant in the original inoculation. However hydrogenotrophic methanogens increase with the improvement of organic load rate,which is conducive to the further degradation of volatile fatty acids and improves the anaerobic digestion performance.