Objective We reported that the expression of heat shock protein 90α (Hsp90α) in the blood of patients with active pulmonary tuberculosis (ATB) and analyze the diagnostic significance in distinguishing active pulmonary tuberculosis from latent pulmonary tuberculosis. Methods In this study, 81 patients with ATB and 65 patients with latent tuberculosis (LTBI) diagnosed in the pulmonary tuberculosis area of Anhui Provincial Hospital Infection Hospital from January 2021 to June 2021 were selected to observe and compare the plasma samples of the two groups of patients. The Hsp90α expression at ATB has been observed, the receiver operating characteristic (ROC) was used to analyze the sensitivity and specificity of the diagnosis of ATB, and the diagnostic efficacy (sensitivity and specificity) of Hsp90α and T-cell enzyme-linked immunospot test (T-spot.TB) was compared. Results Hsp90α in plasma was over represented at the ATB group and higher than that in the LTBI group, with statistically significant difference (P＜0.05). Its sensitivity and specificity for differentiating ATB and LTBI were 74.1% and 81.8% raspectively; The area under the ROC curve was 0.745 (95% CI: 66.4% to 82.6%). The susceptibility and atopic of T-spot.TB for distinguishing active and latent tuberculosis were 40.7% and 84.4%, and the area under the ROC curve was 0.624 (95%CI: 0.536~0.712). Conclusion After Mycobacterium tuberculosis infection, Hsp90α in plasma will be up-regulated, and the positive rate and expression level of ATB are higher than those of LTBI. The sensitivity and specificity of quantitative detection of Hsp90α are higher than T-spot.TB. in diagnasing ATB. Hsp90α is expected to be a new biomarker option to aid in the diagnosis of ATB