This study was performed aiming to evaluate the sensitivity of T-SPOT.TB assay in diagnosis of pediatric TB and investigate the association between age and results of T-SPOT.TB assay. Between March 2012 and September 2013, 102 patients, who aged ≤15 years old, were enrolled in this retrospective study and then were grouped into three age categories: Group 1, aged ≤2 years; Group 2, aged 3 to 12 years; and Group 3, aged 13 to 15 years. The χ² test was used to compare the sensitivities of T-SPOT.TB between groups with different ages. The overall sensitivity of T-SPOT.TB assay was 58.8% (95% confidence interval: 49.1–67.9%) and low in diagnosis of pediatric TB. Although the sensitivities varied in the three groups, the difference did not reach statistical significance (p > 0.05). Therefore, in high burden countries, T-SPOT.TB assay wasn't accurate in diagnosis of pediatric TB, and age was not associated with the results of T-SPOT.TB