Abstract: Objective To establish a effective and rapid method by Ethidium Monoazide Bromide(EMA) in combination with PCR(EMA-PCR) for thedetection of live Pseudomonas aeruginosa. Methods The oprI gene was used as the target gene for PCR detection of Pseudomonas aeruginosa, and PCR amplification was carried out by utilizing its pure isolates as the template. Sensitivity, EMA concentration and exposure time were optimized. Results The sensitivity of PCR detection was 3×103 CFU/mL, exposure time was 10 min. when the EMA concentration was not more than 5 μg/mL, no obvious inhibition to the amplification of DNA derived from viable bacteria was observed. The PCR amplification of DNA derived from 3×106 CFU/mL dead cells could be inhibited effectively by EMA at the final concentration of 1 μg/mL. The results demonstrated the establised method could detect 1% live bacteria from a mixed bacterial population. Conclusion EMA-PCR can detect live bacteria of Pseudomonas aeruginosa effectively and avoid false positive result of the PCR detection.