Abstract: Objective To establish a single gene-based assay of real time fluorescent quantitative reverse transcription polymerase chain reaction (rRT-PCR) assay to detect Salmonella (S.) Choleraesuis. Methods To obtain the specific genes of S. Choleraesuis, two screening rounds were performed. Firstly, the genes shared by sequenced S. Choleraesuis strains were identified and aligned with the genome sequences of other Salmonella serotypes in GenBank, and with the human genome. The alignments less than 50% of the gene size were ignored. In the second round, 123Salmonella strains covering 24Salmonella serotypes were used to examine the specificity of these genes by PCR. The result specific gene, SC1242, was used as the target to develop the rRT-PCR assay. The specificity and detection limit of the rRT-PCR assay was evaluated by using pure cultured strain and S. Choleraesuis simulated blood specimens. Results Twenty S. Choleraesuis isolates were amplified to be positive with established rRT-PCR assay, other 103 isolates were amplified to be negative. For purified total RNA from the pure cultured isolates, the detection limit of the assay was 50 fg/l per reaction, equal to 96 molecular copies per reaction. The sensitivity was 90 cfu/ml in the extraction of nucleotide from the simulated blood. Conclusion The established rRT-PCR assay for detecting S. Choleraesuis with high sensitivity and specificity would be suitable for the rapid detection of S. Choleraesuis.