Objective To explore the efficacy of T-cell spot test of tuberculosis infection (T-SPOT. TB) in the differential diagnosis of spinal tuberculosis (STB), and optimize diagnostic efficacy through the optimal cut-off value of receiver operating characteristic (ROC) curve. Methods Clinical data of patients with spinal infection in a hospital from January 2010 to May 2019 were collected, including preoperative T-SPOT. TB test results, white blood cell count, C-reactive protein, erythrocyte sedimentation rate, procalcitonin, and tuberculosis antibodies, et al. Clinical diagnosis was conducted based on diagnostic criteria. The sensitivity and specificity of T-SPOT. TB in preoperative diagnosis of STB and other spinal infection was analyzed, and the diagnostic efficacy of the optimized T-SPOT. TB indicators was evaluated. Results A total of 132 patients were included in this study, out of whom 78 patients (59.09%) were diagnosed with STB, and 54 (40.91%) were diagnosed with non-tuberculosis (non-TB) spinal infection. The sensitivity and specificity of T-SPOT. TB in differential diagnosis of STB were 67.68% and 66.67%, respectively. Univariate logistic regression analysis showed that compared with non-TB spinal infection, the OR va-lue of T-SPOT. TB test in diagnosing STB was 4.188 (95%CI: 1.847-9.974, P < 0.001). The optimized T-SPOT. TB evaluation index through ROC curve to determine the optimal cut-off values of ESAT-6, CFP-10, and CFP-10+ESAT-6 for differential diagnosis of STB and non-TB spinal infection were 12.5, 19.5, and 36, respectively, and area under curve (AUC) values were 0.765 6, 0.741 5, and 0.778 6, respectively, all with good diagnostic efficacy. CFP-10+ESAT-6 had the highest AUC. CFP-10+ESAT-6 specific spot count had higher efficacy in the diagnosis of STB, with a diagnostic accuracy of 75.56%, higher than 67.42% of pre-optimized T-SPOT. TB. Conclusion T-SPOT. TB test has high diagnostic efficacy in differentiating STB from non-TB spinal infection. Positivity in T-SPOT. TB test, especially with spot count of CFP-10+ESAT-6 over 36, indicates a higher likelihood of STB.