Abstract: Objective To develop and optimize real-time florescence quantitative PCR (FQ-PCR) with the housekeeping gene RAG2 as cell number control to quantify T-cell receptor excision circle (TREC) in the peripheral blood. Methods The real-time PCR system for amplifying TREC and RAG2 genes was established on the basis of ABI 7000 apparatus using Golden Taq system, designed primers, TaqMan-MGB probes and optimized buffer. PCR conditions were optimized with standard samples of TREC plasmid. Results The amplification with the primer pair T3 and T4 was more efficient than that with T1 and T2. More specific and efficient amplification in FQ-PCR was achieved using TaqMan-MGB probes as compared with general Taq-Man probes. Golden Taq was more effective than general Taq in improving the specificity and decreasing the artifact, and 95 ℃ for 10 min, 95 ℃ for 5 s, and 53 ℃ for 30 s for a total of 40 cycles using ABI7000 was found as the optimized thermal parameter setting. Conclusion An optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established.