Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of �16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.