SETTING: An important limitation of the T-SPOT (R) TB assay is its inability to distinguish active tuberculosis (TB) from latent tuberculous infection (LTBI). OBJECTIVE: We proposed a new calculation method for the T-SPOT assay and assessed its effect on distinguishing active TB from LTBI. DESIGN: A total of 162 active TB patients and 97 LTBI individuals were diagnosed according to conventional tests and the T-SPOT assay. RESULTS: The results of early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in T-SPOT cannot be recommended for distinguishing TB from LTBI. The number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in the T-SPOT assay was reduced in active TB patients. The ESAT-6/PHA or CFP-10/PHA ratios in active TB patients were significantly higher than in individuals with LTBI. Using 0.295 as the threshold ratio of Mycobacterium tuberculosis-specific antigen (TBAg) sfc to PHA sfc (TBAg/PHA ratio, the larger of ESAT-6/PHA and CFP-10/PHA), the sensitivity and specificity were 82.1% and 90.7% in distinguishing active TB from LTBI. The TBAg/PHA ratio might also be used to monitor the effect of anti-tuberculosis treatment. CONCLUSIONS: Calculating the TBAg/PHA ratio might have the potential to diagnose active TB and distinguish TB disease from LTBI.