Abstract: In activated sludge process the separation of sludge and water is achieved by gravity, and the floc-forming bacteria are predominated and play a central role in the water purification. In this study, a floc-forming bacterial strain YN12 was isolated from activated sludge of the municipal sewage treatment plant of Luxi County, Yunnan Province, and it was identified as Pseudoduganella eburnean and was phenotypically similar to the type strain 10R5-21^T. In order to reveal and compare the floc-forming mechanism and carbon source utilization of the strain YN12 with those of other floc-forming bacteria, the genome sequencing, assembly, annotation and comparative genomics analysis were conducted. The results showed that the genome size of Pseudoduganella eburnea YN12 was about 5934 kb, the G+C content was 63.9%, and it contained 5313 protein coding sequences. Comparative analyses revealed a similar floc-forming and regulation mechanism to Zoogloea resiniphila MMB, Aquincola tertiaricarbonis RN12 and Mitsuaria chitosanitabida XHY-A6, which involves the exopolysaccharide biosynthesis, PrsK-PrsR two-component system and PEP-CTERM domain containing protein family. Meanwhile, these genes involved in the synthesis of extracellular polymeric substances (EPS) were concentrated on a large gene cluster of about 72 kb in size, and the YN12 strain was more functionally diverse in the absorption and utilization of carbon sources, particularly monosaccharides, disaccharides and polysaccharides. Moreover, two other Pseudoduganella eburnea strains had also been isolated from a river and an aquaculture pond. It is suggested that these strains could also be utilized in biofloc technology to improve the water quality and nutrient recycling in aquaculture.