We compared the positive rates of T-SPOT.TB and bacterial culture in the smear-negative PTB, and analyzed the factors affecting the results of negative T-SPOT.TB and bacterial culture. Retrospective evaluation of data from smear-negative PTB patients who underwent T-SPOT.TB and bacterial culture were done. The agreement and concordance were analyzed between T-SPOT.TB and bacterial culture. Multivariable logistic regression analysis was used to explore the factors associated with positive results of T-SPOT.TB and bacterial culture in smear-negative PTB. 858 eligible smear-negative PTB patients were included in the study. The agreement rate was 25.6% (22.7~28.5%) between T-SPOT.TB and bacterial culture in smear- negative PTB patients. The positive rate of T-SPOT.TB was higher than that of bacterial culture in smear-negative PTB patients (p < 0.001). There were nearly no concordance between T-SPOT.TB and bacterial culture (p > 0.05). Using multivariable logistic regression analysis we found that older age ≥ 60 years (OR = 0.469, 95% CI: 0.287-0.768) and decreased albumin (OR = 0.614, 95% CI: 0.380-0.992) were associated with negative diagnostic results of T-SPOT.TB in smear-negative PTB patients. Female (OR = 0.654, 95% CI: 0.431-0.992) were associated with negative diagnostic results of bacteria culture in smear-negative PTB patients. Our results indicated that the older age and decreased albumin were independently associated with negative T-SPOT.TB responses.