Objective To analyze the prevalence of latent tuberculosis infection (LTBI) among relevant healthcare workers (HCWs)in designated tuberculosis medical institutions (MIs) in Yantai City, and explore its influencing factors. Methods The cluster random sampling method was adopted to select two county- and district-level designated tuberculosis MIs. All HCWs underwent questionnaire survey and creation tuberculin skin test (C-TST) at the same time, and the influencing factors for LTBI were analyzed. Results A total of 215 HCWs from designated tuberculosis MIs were included for analysis, 37 were diagnosed with LTBI, with an infection rate of 17.21% (95%CI: 12.42%-22.93%). Multivariate logistic regression analysis showed that clinicians (OR=3.19, 95%CI: 1.05-9.69), laboratory technician (OR=5.90, 95%CI: 1.21-28.77), working years ≥10 years (OR=3.31, 95%CI: 1.39-7.90), and tuberculosis history of family members (OR=6.49, 95%CI: 1.01-41.46) were independent risk factors for LTBI. Conclusion The infection risk of clinicians and laboratory technicians who directly contact with tuberculosis patients or Mycobacterium tuberculosis is higher than that of other HCWs, and is related to the length of working years. It is suggested that healthcare-associated infection control measures should be streng-thened, and tuberculosis active screening should be carried out regularly for HCWs in key departments.