Background: This study aimed to establish and evaluate a simultaneous amplification and testing method for detection of extra-pulmonary tuberculosis. Methods: From January 2016 and December 2017 the pus or surgical excision from the lesions of inpatients admitted from Chongqing Public Health Treatment Center were collected. According to the clinical diagnosis, the samples were divided into two groups including extra-pulmonary tuberculosis (Group A) and other diseases excluded from tuberculosis diseases (Group B). Simultaneous detection of Mycobacterium tuberculosis (MTB) used Roche culture method, liquid culture method and simultaneous amplification and testing method (SAT). The sensitivity and specificity of the SAT method were compared with culture methods and clinical diagnosis of extra-pulmonary tuberculosis. Results: For 433 extra-pulmonary tuberculosis (EPTB) specimens and 49 non-TB specimens, the SAT-TB results correlated with 80.5% (388/482 specimens) of the culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of EPTB were 83.6%, 79.4%, 59.4%, and 93.0%, respectively, compared to culture methods. Compared with the clinical diagnosis of patients, the sensitivity and specificity of the SAT-TB test were 41.6% and 100%, respectively, the cultures test were 29.3% and 98.0%. Conclusions: SAT test is a simple and rapid test with high specificity which may enhance the detection of EPTB. SAT-TB is a higher clinical diagnosis value for EPTB in clinical microbiology laboratories.