Chagas disease is caused by the parasite . In humans, it evolves into a chronic disease, eventually resulting in cardiac, digestive, and/or neurological disorders. In the present study, we characterized a novel antigen named Tc323 (TcCLB.504087.20), recognized by a single-chain monoclonal antibody (scFv 6B6) isolated from the B cells of patients with cardiomyopathy related to chronic Chagas disease. Tc323, a ~323 kDa protein, is an uncharacterized protein showing putative quinoprotein alcohol dehydrogenase-like domains. A computational molecular docking study revealed that the scFv 6B6 binds to an internal domain of Tc323. Immunofluorescence microscopy and Western Blot showed that Tc323 is expressed in the main developmental forms of , localized intracellularly and exhibiting a membrane-associated pattern. According to phylogenetic analysis, Tc323 is highly conserved throughout evolution in all the lineages of so far identified, but it is absent in spp. and . Most interestingly, only plasma samples from patients infected with and those with mixed infection with spp. reacted against Tc323. Collectively, our findings demonstrate that Tc323 is a promising candidate for the differential serodiagnosis of chronic Chagas disease in areas where and spp. infections coexist.