Background: Discriminating active tuberculosis (ATB) from latent tuberculosis infection (LTBI) remains challenging. This study aimed to investigate a diagnostic model based on a combination of iron metabolism and the TB-specific antigen/phytohemagglutinin ratio (TBAg/PHA ratio) in T-SPOT.TB assay for differentiation between ATB and LTBI. Methods: A total of 345 participants with ATB (n = 191) and LTBI (n = 154) were recruited based on positive T-SPOT.TB results at Tongji hospital between January 2017 and January 2020. Iron metabolism analysis was performed simultaneously. A diagnostic model for distinguishing ATB from LTBI was established according to multivariate logistic regression. Results: The TBAg/PHA ratio showed 64.00% sensitivity and 90.10% specificity in distinguishing ATB from LTBI when a threshold of 0.22 was used. All iron metabolism biomarkers in the ATB group were significantly different from those in the LTBI group. Specifically, serum ferritin and soluble transferrin receptor in ATB were significantly higher than LTBI. On the contrary, serum iron, transferrin, total iron binding capacity, and unsaturated iron binding capacity in ATB were significantly lower than LTBI. The combination of iron metabolism indicators accurately predicted 60.00% of ATB cases and 91.09% of LTBI subjects, respectively. Moreover, the combination of iron metabolism indexes and TBAg/PHA ratio resulted in a sensitivity of 88.80% and specificity of 90.10%. Furthermore, the performance of models established in the Qiaokou cohort was confirmed in the Caidian cohort. Conclusions: The data suggest that the combination of iron metabolism indexes and TBAg/PHA ratio could serve as a biomarker to distinguish ATB from LTBI in T-SPOT-positive individuals. (C) 2020 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).