Based on the sewage treatment device of Two-phase Integrated Sludge Thickening and Digestion reactor (TISTD), microbial community structure in stable operation and its ecological variation when load changes are investigated. During a stable operation with 30% sludge dosage rate, 20 dominant bacterial strains are individually isolated from acid-phase and methane-phase of the reactor. Sequence analysis of 16S rDNA reveals that six of the achieved strains belong to Bacillus, Methanospirillum and Methanococcus respectively. Ten sludge samples are collected separately in 5 periods when the reactor operates with 10%, 20% and 30% sludge dosage rate and in the state of started-up and disordered, and followed by DNA extraction, purification, 16S rDNA PCR amplification and temperature gradient gel electrophoresis (TGGE). The good quality of DNA extraction and amplification demonstrates that there is an abundant biomass in the sludge under mid-temperature condition. The results show that TISTD reactor has a high biodiversity in microbial communities and is complex and stable in ecosystem structure, which effectively supports the good digestion in sludge thickening.