This study aimed to establish human IFN-gamma (hIFN-gamma) in vitro release assay and to apply it in diagnosis of human tuberculosis. Human IFN-gamma gene was cloned and expressed in Escherichia coli. The recombinant hIFN-gamma was purified and used as immunogen to immunize mice and rabbits respectively. Monoclonal and polyclonal antibodies were respectively developed and a sandwich ELISA was established. The heparized whole blood from 111 active tuberculosis patients and 292 clinical healthy controls were collected. The blood was stimulated with tuberculosis specific fused antigen ESAT-6/CFP-10 and the plasma was collected for IFN-gamma detection. The sensitivity for tuberculosis diagnosis was 95.5%, whereas the positive detection rate for the healthy controls was 16.7%. There was a significant difference between the patients and healthy controls (P<0.01) indicating that this assay had a high sensitivity and specificity, and thus could be promising in tuberculosis diagnosis.