Objective To identify Mycobacterium tuberculosis ESAT-6 protein esxN-specific HLA-A*0201-restricted CTL epitopes and assess the diagnostic potential of the identified epitopes in pulmonary tuberculosis. Methods The esxN-specific HLA-A*0201-restricted CTL epitopes were predicted by the T epitope prediction software SYFPEITHI and further synthesized. The binding affinity of the candidate epitopes for HLA-A*0201 was detected using MHC-peptide complex stabilization assay. The immunogenicity of candidate epitopes were assessed using ELISPOT in HLA-A*0201 transgenic mice. Based on identified CTL epitopes, ESAT-6 and culture filtrate protein-10 (CFP-10), the ELISPOT was performed to detect the frequency of epitope/protein-specific CTL. Results In six CTL epitope candidates we tested, two epitopes, esxN15-24 (AMIRAQAASL) and esxN48-57 (VACQEFITQL), were found to have a high affinity for HLA-A*0201. In the HLA-A*0201 transgenic mice immunized with the epitope candidates, esxN48-57 induced T-cell response with a significantly high IFN-γ secretion. The IFN-γ-producing T cells directed to esxN15-24 and esxN48-57 were found to be correlated with the presence of ESAT-6 and CFP-10 in positive pulmonary tuberculosis patients. The sensitivity of these tests for the esxN15-24 and esxN48-57 epitopes was similar to that of ESAT-6 and CFP-10. Conclusion Two novel Mycobacterium tuberculosis protein esxN-derived HLA-A*0201-restricted CTL epitopes have potential for the diagnosis of tuberculosis.