Mycobacterium tuberculosis (MTB)-specific antigens, ESAT-6 or CFP-10 play a key role in diagnosis and control MTB infection. T cell receptor (TCR) reflects the status and function of T cells. However, the features of the TCR beta variable (TCRBV) repertoire used against ESAT-6 and CFP-10 from MTB subjects have not been well described. The molecular profiles of TCRBV complementarity-determining region 3 (CDR3) in PBMCs with or without ESAT-6 or CFP-10 stimulation were assayed using a gene melting spectral pattern (GMSP) assay developed in our previous study. The average number of skewed TCRBV family in PBMCs stimulated with ESAT-6 or CFP-10 was significantly higher than that in unstimulated PBMCs. TCRBV3, BV5.1, BV12, BV13.1, BV13.2, BV20 and BV24 were used more frequently than other TCRBV members in PBMCs from MTB subjects, and TCRBV3, BV5.1 in stimulated PBMCs have a preference in the usage of variable (V) and joining (J) segments and CDR3. The results indicate that the T cell immune response in MTB subjects involves a few of specific T cells. The preferred usage of certain V and J segments and CDR3s of TCRBV3 or BV5.1 may be related to ESAT-6 or CFP-10 respectively, which would help clinical differential diagnosis and treatment of MTB-infected subjects.