Extrapulmonary tuberculosis (EPTB) has become more common in recent years; however, the diagnosis of EPTB remains a challenge. In this study, we analyzed the performance of the ratio of TB-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) in T-SPOT. TB (T-SPOT) assay for diagnosis and treatment monitoring of EPTB. Between 2012 and 2017, 734 EPTB patients were diagnosed and recruited from Tongji hospital, and 1,137 suspected EPTB patients who had other diagnoses were recruited as non-EPTB controls. To validate the study, another small group of EPTB patients and non-EPTB controls were recruited from Sino-French New City Branch of Tongji Hospital. The positive rate of peripheral blood T-SPOT in EPTB and non-EPTB were 88.15 and 32.28%. In T-SPOT positive patients, the direct T-SPOT results had limited value in distinguishing these two conditions. A further calculation of the TBAg/ PHA ratio of T-SPOT showed improved performance in each form of EPTB. If using 0.20 as the threshold value of the TBAg/PHA ratio, the pooled sensitivity and specificity were 70.79 and 91.55% in distinguishing EPTB from non-EPTB. The validation results showed a better performance of the TBAg/PHA ratio in distinguishing these two conditions, with a sensitivity and specificity of 81.82 and 97.56%, respectively. Comparing with directly using T-SPOT results, the TBAg/PHA ratio was less affected by immunosuppression. Furthermore, PHA value reflected immunosuppression and could help to judge the credibility of T-SPOT results in EPTB patients with different immune status. The TBAg/PHA ratio was significantly decreased during anti-tuberculosis (TB) treatment, which suggests that it can also be used to monitor therapeutic efficacy. These data provide new insights into the role of T-SPOT assay in TB disease, and the TBAg/PHA ratio might be a useful tool for diagnosis and treatment monitoring of EPTB.