To analyze the effect of DO and aeration/non-aeration ratio on the microbial community structure, active sludge and biofilm samples were taken from the SBBR one-step completely autotrophic nitrogen removal reactor under different DO concentrations and aeration/non-aeration ratios. By PCR-DGGE and bands statistical analyses, results show that through one and a half year of operation, the microbial community structures in this system has changed a lot compared with the inoculum. The bacterial richness values of biofilm are higher than active sludge in 2 h:2 h aeration/non-aeration ratio, no matter when moderate, high and low DO concentration. When this system is operated under the most efficient condition: DO = 2.0 (aeration) mg/L, 0.4 (non-aeration) mg/L, the bacterial richness values are the highest. There are about 14 and 10 bands in the biofilm and active sludge samples respectively. The diversities of bacterium and their co-metabolisms are the key factors of maintaining a high operate efficiency. In addition, the aeration/non-aeration ratio also has the influence on the microbial community structure. Under long aeration/non-aeration ratio of 3 h:5 h, microbial community structure of biofiln is similar with active sludge. The comparability is 100%. The activities of aerobic and anaerobic bacterium have lost much that makes low efficiency of this system although they could survive both in active sludge and biofim.