Abstract: Two-phase anaerobic digestion is one of the most important techniques for organic solid waste treatment. A series of experiments were carried out to determin the effect of innoculum、PH、aeration on the performance of rice straw in two-phase digestion and the biogas production of rice straw between single-phas and two-phase digestion was compared in this paper. The results showed that compared to single-phase system, the average methane content was improved from 50% to 66% in two-phase system, while it has no significant difference on the biogas production of rice straw under the laboratory conditions of 35°C and total solids (TS) content of 8%; Coprinus comatus and anaerobic sludge inoculation could increased the rate of hydrolysis of rice straw, the hemicellulose and cellulose degradations were increased by 2.11 times and 8.92 times respectively, compared to the normal treatment; Intermittent aeration resulted in falling of biogas production, and it had no effect on phase separation; adjusting pH value to 6.0 could effectively inhibit the biogas production in the hydrolysis phase, the ratio of biogas production in hydrolysis phase was reduced from 79.52% to 18.88%, while the ratio of biogas production in methanogenic reactor was increased from 20.48% to 81.12%. The study can provide references for the technology of two-phase anaerobic digestion