To investigate the structural characteristics of microbial community in the anaerobic digestion system of straw and sludge, the Illumina HiSeq high-throughput sequencing technology was used to study the changes and diversity of microbial community structure under high and low substrate loading conditions, and monitor its gas production. Results show that the average daily gas production was about 4.1 L under high load conditions (20 g/(L·d)) and about 2.1 L under low load conditions (12 g/(L·d)). The relative abundance of bacteria under high load substrate conditions was 91.57%, and the relative abundance of methanogenic archaea was 8.43%; the relative abundance of bacteria under low load substrate conditions was 94.35%, and the relative abundance of methanogenic archaea was 5.65%. The relative abundance of methanogenic archaea under high load substrate conditions increased about 49.2% compared with that under low load substrate conditions, indicating that there was a positive correlation between the relative abundance of archaea and gas production. The first three dominant phyla under high load conditions were Bacteroidetes with a relative abundance of 51.06%, Firmicutes with that of 11.65%, and Euryarchaeota with that of 8.25%. The first three dominant phyla under low load conditions were Bacteroidetes with a relative abundance of 50.78%, Cloacimonetes with that of 7.67%, and Synergistetes with that of 6.46%. Twelve kinds of methanogens archaea were detected under the two substrate conditions, which belong to hydrogenotrophic, methylotrophic, and aciticlasitic, indicating that the metabolic pathway of the whole straw and sludge anaerobic digestion system was rich, the growth metabolism of various microbial communities was interdependent and balanced with certain anti-shock loading, and a relatively balanced buffer system was established.