Abstract: Objective To evaluate the usefulness of enzyme-link immunospot assay(ELISPOT) in the diagnosis of sputum smear-negative tuberculosis.Methods Totally 30 tuberculosis patients with sputum smear-positive,106 tuberculosis patients with sputum smear-negative,and 71 non-tuberculosis patients were enrolled.The interferon-gamma(IFN-γ) produced by peripheral blood mononuclear cells of all participants was detected by ELISPOT,and parts of participants were performed with tuberculin skin test(TST) simultaneously.Results The sensitivity of ELISPOT in patients with sputum smear-positive(96.7%,29/30) was higher than that of patients with sputum smear-negative(88.7%,94/106),without significant difference(χ²=0.925,P＞0.05).The sensitivity of TST in patients with sputum smear-positive(78.6%,11/14) were higher than that of patients with sputum smear-negative(65.7%,69/105) with no significant difference(χ²=0.925,P＞0.05).In patients with sputum smear-negative,the specificity of ELISPOT was higher than that of TST with significant difference(χ²=15.83,P＜0.001).However,in control group,the sensitivity of ELISPOT(84.5%,60/71) was higher than that of TST(59.2%,42/71) with significant difference(χ²=11.28,P=0.001),and the specificity of ELISPOT in controls with Bacille Calmette Guerin(BCG) vaccination(73.3%,33/45) was higher than that of patients without BCG vaccination with no significant difference(P＞0.05),while the specificity of TST in controls with BCG vaccination(38.5%,10/26) was lower than that of patients without BCG vaccination(71.1%,32/45) with significant difference(P=0.007).Conclusion The ELISPOT is an supplementary mean for the diagnosis of sputum smear-negative tuberculosis in clinical practice.It would be helpful for the rapid diagnosis and treatment for the patients.