Objective To investigate the diagnostic value of the T cell spot (T-SPOT.TB) test, oxidation-related factors (ORF), and antimicrobial peptide LL-37 in patients with pulmonary tuberculosis (PTB) with type 2 diabetes. Methods A total of 560 patients with PTB admitted to our hospital from January 2019 to April 2021 were retrospectively included in this study. Of these, 232 patients with PTB and type 2 diabetes were assigned to the combined group, and 328 patients without complications were assigned to the PTB group. Results Areas under the curve (AUCs) for the number of spot-forming cells in CFP10 and ESAT-6 test panels detecting PTB with type 2 diabetes were 0.892 (95% confidence interval [CI] 0.831–0.921) and 0.893 (95% CI 0.841–0.935), respectively. CFP10 combined with ESAT-6 had the highest diagnostic value, with sensitivity and specificity levels and an AUC of 87.73%, 88.93%, and 0.942 (95% CI 0.907–0.967), respectively. The levels of total antioxidant capacity, superoxide dismutase, and catalase in the combined group were lower than in PTB and control groups. Conclusion The combination of T-SPOT.TB, ORF, and LL-37 in the diagnosis of pulmonary tuberculosis with type 2 diabetes mellitus has a high diagnostic value and clinical application value.