Objective To construct reference standards for detection and quantification of Klebsiella pneumoniae (K.pneumoniae) with SYBR Green I-based real-time PCR assay. Methods Primers were designed based on the published sequence of the phoE gene of K.pneumoniae. The standard was prepared by cell culture, PCR and T-A clone methods, and was identified by colony PCR and DNA sequencing. Results The standard curve showed a very good linear negative regression between threshold cycle (Ct) and Log starting quantity of copy number. The detection range was from 5.2 to 5.2×106 copies per reaction, and the detection limit was 6 copies per reaction. The coefficients of variance (CVs) of three parallel experiments were in the range of 0.05%-0.91%. Conclusion The reference standards have high stability and reproducibility. They can be used in the quantitative detection of K.pneumoniae.