This study aimed to reveal the associated risk factors for latent tuberculosis infection (LTBI) detected by T-SPOT.TB assay among health care workers (HCWs) at different working locations or job categories in China. This cross-sectional study included 934 HCWs who underwent the T-SPOT.TB assay. Demographic and social characteristics of the participants, including age, sex, job categories, department/ward and duration of healthcare service, were recorded. Among 934 HCWs, 267 (28.5867%) were diagnosed as having LTBI with positive T-SPOT.TB assay. HCWs working in inpatient tuberculosis (TB) (odds ratio (OR) 2.917; 95% confidence interval (CI) 1.852–4.596; P < 0.001) and respiratory wards (OR 1.840; 95% CI 1.124–3.011; P = 0.015), and with longer duration of healthcare service (OR 1.048; 95% CI 1.016–1.080; P = 0.003) were risk factors for positive T-SPOT.TB result. Furthermore, longer working duration increased the positive rate of T-SPOT.TB results for physicians and nurses, and physicians had higher risks than nurses for the same working duration. Inpatient TB and respiratory wards were high-risk working locations for HCWs with LTBI, and longer duration of healthcare service also increased the risk of LTBI among HCWs. A complete strategy for TB infection control and protection awareness among HCWs should be enhanced.