Objective To evaluate the application value of combined detection of Mycobacterium tuberculosis-specific cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2) in the differential diagnosis of pulmonary tuberculosis and bacterial pneumonia. Methods A total of 91 hospitalized patients in the respiratory department of Fuyang People’s Hospital from January 2022 to October 2023 were selected. Forty-five patients were diagnosed with pulmonary tuberculosis (pulmonary tuberculosis group), and 46 patients were diagnosed with bacterial pneumonia (pneumonia group). Two-cytokine combined detection were performed on all patients. The effect of two-cytokine combined detection and C-reactive protein (CRP) in the differential diagnosis of pulmonary tuberculosis and bacterial pneumonia was compared. Results The sensitivity and specificity of two-cytokine combined detection for the differential diagnosis of pulmonary tuberculosis and bacterial pneumonia were 86.7% and 84.8%, respectively. The area under the receiver operating characteristic curve was 0.928 (95%CI: 0.870-0.986), which showed significant difference compared with differential diagnostic efficacy of CRP (P<0.05). Conclusion The combined detection of Mycobacterium tuberculosis-specific cytokine IFN-γ and IL-2 in the differential diagnosis of pulmonary tuberculosis and bacterial pneumonia has a high application value, which can provide a basis for the differential diagnosis of pulmonary tuberculosis and bacterial pneumonia.