Objective: To evaluate clinical application of MTB secreted protein colloidal gold method in diagnosis of pulmonary tuberculosis using sputum sample. Methods: A total of 173 cases of hospitalized tuberculosis patients diagnosed in Binzhou Central Hospital from October 2020 to December 2020 were selected as research objects, and morning sputum was collected and detected by acid-fast staining smear microscopy, sputum culture, MTB secreted protein colloidal gold method and GeneXpert MTB/RIF method, respectively. Diagnostic efficacy was compared in different groups. Results: Among the samples from 173 patients, 59 (34.10 %) were positive by sputum smear, 66 (38.15 %) were positive by sputum culture, 90 (52.02 %) were positive by colloidal gold, and 102 (58.96 %) were positive by GeneXpert MTB/RIF method. There was significant difference between sputum colloidal gold method and sputum culture results (χ²=6.724, P=0.010); the results of GeneXpert MTB/RIF method and sputum culture method were significantly different (χ²=14.995, P＜0.001); there was no significant difference between the results of sputum colloidal gold method and GeneXpert MTB/RIF method (χ²=1.685, P=0.194). The sputum samples of 173 patients was detected, and the positive rate of the bacteria positive group was significantly higher than bacteria negative group(χ²=45.146, P＜0.001;χ²=49.406, P＜0.001). By the sputum colloidal gold method, there was a statistically significant difference between bacteria positive group and bacteria negative group (χ²=45.146, P＜0.001), but there was no significant difference between initial treatment and retreatment (χ²=0.160, P=0.689). Conclusion: MTB secreted protein colloidal gold method has promising clinical value in tuberculosis diagnosis, which is rapid, simple and worth for clinical application widely.