PCR-DGGE was applied to analyze the microbial communities in rotating drum biofilter (RDB) for nitric oxide denitrifying removal under anaerobic conditions. The 16S rRNA genes (V3 region) were amplified with two universal primers (338F-GC and 518R). These amplified DNA fragments were separated by parallel DGGE. The DGGE profiles of biofilm samples from different sections of RDB are similar and about sixteen types of microorganisms are identified in the biofilm samples. These results show that microbial diversity of RDB firstly increases but then decreases with the addition of Cu II (EDTA) and the increase of NO removal efficiency. However, the change of microbial community is not significant during the process. Eight major bands of 16S rRNA genes fragments from DGGE profiles of biofilm samples were further eluted from gel, re-amplified, cloned and sequenced. The sequences of these fragments were compared with the database in GeneBank (NCBI). The gene analysis of 16S rRNA showed that the major populations are Cytopahga-Flexibacteria-Bacteroides (CFB) group Bacteroides, beta -Proteobacterium, gamma-Proteobacterium and Clostridium sp.. In addition, denitrification is related with band G-5, G-6 and G-8. G-5 is affiliated with gamma-Proteobacterium. Both G-6 and G-8 are affiliated with beta-Proteobacterium.