According to the partially known sequences of Escherichia coli，Klebsiella pneumoniae and Acinetobacter in GenBank，the uidA gene of Escherichia coli，khe gene of Klebsiella pneumoniae and secE gene of Acinetobacter were selected. Then 3 pairs of primers and Taqman probes of which 5' end were labeled with fluorophores（FAM，HEX，CY5）respectively ，and 3' end marked with BHQ1 quenchers were designed by Primer Premier 5. By optimizing the reaction conditions，a diagnostic method which could detect Escherichia coli，Klebsiella pneumoniae and Acinetobacter simultaneously was established. Experiments of specificity and sensitivity showed that this method could specifically detect the above three genes，while the lowest detection limits were 2×10-1 CFU/μL，9.2×10-1 CFU/μL and 4.3×10-1 CFU /μL，respectively. The whole amplification was completed within two hours. In conclusion，a triple quantitative PCR with good sensitivity，reproducibility and specificity was established ，it could be used for rapid and simultaneous detection of the three pathogens.