Objective To explore the characteristics of T-cell receptor beta (TCRβ) gene rearrangement in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system for quantitative detection of minimal residual disease (MRD) by real-time quantitative PCR (RQ-PCR) targeting the TCRβ gene rearrangement. Methods Multiplex PCR designed by the BIOMED-2 was used to detect TCRβ gene rearrangement in the bone marrow samples of 26 children with T-ALL. Sequences of junctional region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRβ gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient-specific standard curves. Subsequently, TCRβ RQ-PCR was applied to six patients to quantify MRD with germline Jβ primer/probe combinations. To determine the quantity and quality of DNA, we also used RQ-PCR for the N-ras gene.Results Clonal rearrangements were identified in 92.3% of the children with T-ALL ( Vβ-Dβ-Jβ rearrangements in 84.6% and Dβ-Jβ rearrangements in 50% ). Comparative sequence analysis of 42 TCRβ recombination revealed that two downstream Vβ families (BV5, BV6) were preferentially used. The segment Jβ2. 7 was dominant in childhood T-ALL. Jβ1. 3, Jβ2.4, and Jβ2.6 were not detected. The slope of the standard curves was from - 3.54 to -3.37 with the intercepts between 19.35 and 20.51. The correlation coefficients of all the 6 standard curves were ≥0.98. None of the cases had a quantitative range of RQ-PCR lower than 10