Antigen assay is a better indicator of active infection than antibody detection hence affinity purified anti ES-31 antibody against Mycobacterium tuberculosis H37Ra Excretory-Secretory 31kDa serine protease antigen (SEVA TB ES-31) was explored for detection of circulating free and Immune-Complexed (IC) ES-31 antigen by microtitre plate Peroxidase sandwich ELISA. The assay was evaluated in 27 clinical sera of sputum acid fast bacilli (AFB) positive and 10 AFB negative but anti-tuberculosis therapy responded pulmonary tuberculosis patients in a tertiary hospital and 20 normal sera as control. The assay for circulating free ES-31 antigen in this preliminary study showed a sensitivity of 74% for AFB positive cases and 70% for AFB negative cases with 90% specificity. The assay for IC ES-31 antigen showed sensitivity of 77% for AFB positive and 70% for AFB negative cases with 90% specificity. Detection of IC-antigen as adjunct assay improves the sensitivity of detection. Peroxidase enzyme immuno assay showed a sensitivity of detection of 0.5 μg/ ml and levels of free and IC ES-31 antigens were 0.71 ± 0.64 and 0.82 ± 0.40 μg/ ml in AFB positive patients’ sera.