Objectives: The risk of transmission of Mycobacterium tuberculosis from patients with tuberculosis to health care workers (HCWs) has been well documented but little is known about the prevalence of latent tuberculosis infection (LTBI) in Iranian HCWs. The aim of this study was to determine the prevalence of LTBI among HCWs by using IFNgamma-release assay and compare the results with those of tuberculin skin test (TST). Methods: Two hundred HCWs were evaluated with both TST and QuantiFERON-TB Gold In Tube test (QFT-GIT). The obtained data were analyzed by SPSS v.16 Software. Results: The participants were 73 males and 127 females with the mean age of 34.36±8.26 years. TST was positive in 105 cases (52.5%) and the QFT results were positive in 17 cases (8.5%). There was poor agreement between the two tests (53%, к = 0.115). Induration diameter of TST ≥ 10 mm and working duration ≥ 10 years were independent predictors for positive QFT (p = 0.004). Conclusions: Due to the fact that BCG vaccination has been administered routinely to all HCWs in Iran, specifi c tests should be introduced for high risk groups. QFT thus seems to be more effective for LTBI diagnosis than TST among HCWs with BCG immunization history.