Introduction China has a very high tuberculosis (TB) burden. The interferon-gamma release assay (IGRA) is more specific for the diagnosis of latent tuberculosis infection (LTBI) than the tuberculin skin test, especially among populations with a high degree of coverage by the BCG vaccine. Objectives Methods To evaluate the first screening of healthcare workers (HCW) for LTBI using the IGRA at a general hospital in Beijing. A pilot screening program for LTBI was triggered by accidental contact between HCW and two patients with active TB in the emergency department (ED). Given the necessity of estimating the overall LTBI prevalence in the institution, a sample of 518 HCW was enrolled in our cross-sectional study. The second IGRA was repeated with 43 of the 121 HCW in the ED after exposure to index TB cases. Data on putative risk factors were collected with a self-administered questionnaire. Results Conclusions The prevalence of LTBI in the targeted population was 21.8%. Differences in the prevalence of LTBI were significantly related to age, employment duration, and history of occupational exposure. A lack of childhood BCG vaccination was independently associated with the prevalence of LTBI (adjusted OR: 1.686, 95% CI: 1.045-2.723, P = .0325). No new LTBI was diagnosed 12 weeks postexposure. No HCW adopted the preventive treatment for LTBI. Considering the high morbidity of LTBI among HCW even in general hospitals, it is essential to formulate government policies and institutional operation protocols for the systematic screening, registration, and administration of prophylaxes for the control of LTBI.