Background Serum adenosine deaminase (ADA) measurement is a biochemical test that is believed to play a role in diagnosing tuberculosis (TB). However, few studies have been conducted in this area and there have been contradictory reports on the diagnostic value of serum ADA in TB. Objective The aim of the study was to evaluate the usefulness of ADA measurement in the serum for diagnosing TB. Materials and methods In all, 97 participants were included in this study; 28 with pulmonary tuberculosis (PTB), 20 with extrapulmonary tuberculosis (EPTB), 32 with non-TB lung disease, and 17 heathy individuals. Serum ADA activity was determined in all participants using the semiautomated biochemistry analyzer ERMA AE600N (ERMA Inc.). Results We found that serum ADA levels were significantly high in patients with TB (PTB and EPTB) compared with non-TB lung disease cases and healthy control. The best cutoff value in this study of serum ADA in diagnosing TB was more than 21.1 IU/l for PTB and more than 21.2 IU/l for EPTB as obtained by the receiver operating characteristic curve with high sensitivity and specificity. Conclusion According to the results indicating high sensitivity for serum ADA level, this test is a helpful tool for the diagnosis of TB. On the basis of high specificity for serum ADA level, this test is also a useful test to rule out TB in questionable cases