Biogas production from lignocellulosic hydrolysate is of great potential for lignocellulosic materials. Two-phase anaerobic digestion was proposed in this study. Acidogenic fermentation was carried out with corn straw hydrolysate as feedstock for volatile fatty acids (VFAs) production. Using anaerobic sludge (AnS), different inoculum to substrate ratios (ISRs) of 0.5:1, 1:1 and 2:1 were investigated. The highest VFAs yield was obtained at ISR of 0.5:1.VFAs composition analysis showed that butyric acid was the predominant acid, followed by acetic acid and propionic acid. The effects of AnS and aerobic sludge (AeS) on the acidogenic performance of hydrolysate were compared. The optimum VFA yields were 0.38 g/g COD-added for AnS and 0.32 g/g COD-added for AeS with HRT of 5 d, respectively. The bacterial diversities of inocula and digestates were analyzed by high-throughput sequencing. Two origins of inocula had distinct bacterial structures, but they did share core communities that included Firmicutes, Chloroflexi, Proteobacteria and Bacteroidetes at phylum level. The bacterial communities of both digestates changed significantly as compared with those in inoculum. Firmicutes was absolutely dominant in all the bacterial species. Therefore, the AeS could be an option as the acidogenic inoculum. The microbial information will be beneficial for the enrichment and acclimatization of microbes. In methanogenic process, VFAs obtained in acidogenic stage could be efficiently converted into methane. The ultimate methane yield at organic loading rate (OLR) of 8 g/L·d could reach 290 mL CH/g COD-added and 279 mL CH/g COD-added for AnS and AeS acidified digestate. Two-phase anaerobic digestion was proved to be suitable for bioconversion of lignocellulosic hydrolysate into biogas.