Background: Klebsiella pneumonia carbapenemases (KPCs) are able to hydrolyze the carbapenems, which cause many bacteria resistance to multiple classes of antibiotics, so the rapid dissemination of KPCs is worrisome. Laboratory identification of KPCs-harboring clinical isolates would be a key to limit the spread of the bacteria. This study would evaluate a rapid low-cost real-time PCR assay to detect KPCs. Methods: Real-time PCR assay based on SYBR Greenl was designed to amplify a 106 bp product of the bla(KPC) gene from the 159 clinical Gram-negative isolates resistant to several classes of beta-lactam antibiotics through antimicrobial susceptibility testing. We confirmed the results of real-time PCR assay by the conventional PCR-sequencing. At the same time, KPCs of these clinical isolates were detected by the modified Hodge test (MHT). Then we compared the results of real-time PCR assay with those of MHT from the sensitivity and specificity. Moreover, we evaluated the sensitivity of the real-time PCR assay. Results: The sensitivity and specificity of the results of the real-time PCR assay compared with those of MHT was 29/29(100 %) and 130/130(100 %), respectively. The results of the real-time PCR and the MHT were strongly consistent (Exact Sig. (2-tailed) = 1.000; McNemar test). The real-time PCR detection limit was about 0.8 cfu using clinical isolates. Conclusion: The real-time PCR assay could rapidly and accurately detect KPCs-harboring strains with high analytical sensitivity and specificity.