The present work focused on a rapid enrichment and cultivation of denitrifying phosphate-removal bacteria (DPB) in a membrane bio-reactor(MBR) by using A2/O anaerobic sludge from a wastewater treatment plant as seed, as well as providing an identification method. In the experiments, sodium acetate was used as the carbon source and a certain amount of nitrate was added to the MBR in the anoxic stage. Results showed that, with the efficient trap of the hollow-fiber membrane module, the proportion of DPB in all the phosphate-accumulating organisms (PAOs) increased from 24% to 93% within 35 days after two-stage's cultivation including anaerobic/aerobic and anaerobic/anoxic, during which the removal efficiency of nitrogen and phosphorus reached more than 90%. The activated sludge was identified by combining a regular method and the fluorescence in situ hybridization (FISH) technique, which demonstrated that Pseudomonas sp. and Rhodocyclus sp. were the dominant bacteria in the used bioreactor.