Abstract: Objective To establish a real-time TaqMan polymerase chain reaction (PCR) assay for the detection of Citrobacter freundii. Methods Primers and probe were designed based on the sequences of tricarboxylic transport (tct) gene.The target gene was cloned to pMD20-T vector to build the standard curve of this assay and evaluate the sensitivity of the assay. The specificity was evaluated by using 20 other enteropathogenic bacteria and isolates causing nosocomial infection. Results Sensitivity test of recombinant plasmids showed that the sensitivity could reach 1?101copies /reaction.Specificity test showed that no specific amplifications were presented for the 20 other enteropathogenic bacteria and the isolates causing nosocomial infection. The detection limit of this assay for artificially contaminated milk was 1.0?102cfu/ml. Conclusion This real-time TaqMan PCR assay is sensitive and specific for the rapid detection of Citrobacter freundii.