To develop the SYBR Green I real-time quantitative PCR method for detection of S gene of transmissible gastroenteritis virus (TGEV), a pair of primers was designed based on the S gene of TGEVMiller strain in GenBank, and the cDNA was obtained by reverse transcriptase polymerase chain reaction (RTPCR) from the TGEV vaccine strain. The purified PCR product was cloned into the pGEMT Easy vector. The resulting recombined plasmid was used as the template for developing the standards curve of the SYBR Green I real-time PCR. The results showed that the developed SYBR Green I real-time PCR could detect 30 copy/μL of plasmid DNA with good specificity and repeatability. All the results suggested that the SYBR Green I real-time PCR we established in the current study could be used in clinical diagnosis and in epidemiological investigation for TGEV.