Hemodialysis (HD) patients should be screened for latent tuberculosis (TB) infection. We aimed to determine the frequency of latent TB infection in HD patients and to compare the effectiveness of the tests used. The files of 56 HD patients followed between 1 January 2021 and 1 October 2022 were retrospectively analyzed. Demographic data, the presence of the Bacillus Calmette-Guerin (BCG) vaccine, whether or not the patients had previously received treatment for TB before, the status of encountering a patient with active TB of patients over 18 years of age, without active tuberculosis and who had a T-SPOT.TB test or a Tuberculin Skin Test (TST) were obtained from the patient files. The presence of previous TB in a posterior–anterior (PA) chest X-ray was obtained by evaluating PA chest X-rays taken routinely. Of the patients, 60.7% (n = 34) were male and their mean age was 60.18 ± 14.85 years. The mean duration of dialysis was 6.43 ± 6.03 years, and 76.8% (n = 43) had 2 BCG scars. The T-SPOT.TB test was positive in 32.1% (n = 18). Only 20 patients (35.7%) had a TST and all had negative results. While the mean age of those with positive T-SPOT.TB results was higher (p = 0.003), the time taken to enter HD was shorter (p = 0.029). T-SPOT.TB test positivity was higher in the group that had encountered active TB patients (p = 0.033). However, no significant difference was found between T-SPOT.TB results according to BCG vaccine, albumin, urea and lymphocyte levels. Although T-SPOT.TB test positivity was higher in patients with a previous TB finding in a PA chest X-ray, there was no statistically significant difference (p = 0.093). The applicability of the TST in the diagnosis of latent TB infection in HD patients is difficult and it is likely to give false-negative results. The T-SPOT.TB test is not affected by the BCG vaccine and immunosuppression. Therefore, using the T-SPOT.TB test would be a more appropriate and practical approach in the diagnosis of latent TB in HD patients.