Detection of mammalian tissue in non-mammalian meat or feed products is important for enforcement of food-labeling laws and prevention of the spread of transmissible spongiform encephalopathies (TSEs). This study was conducted to develop a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for rapid detection of raw, cooked (100 degree Celsius, 30 min) and autoclaved (121 degree Celsius/1.2 bar, 30 min) mammalian meats (beef, deer, elk, horse, lamb and pork) adulterated in non-mammalian meat (chicken, duck and turkey) and soy-based feed products, and to assess the performance of the assay. This assay utilized a pair of MAbs against thermal-stable skeletal muscle protein, troponin I (sTnI). MAb 6G1, specific to mammalian and poultry sTnIs, was used as the capture antibody and horseradish peroxidase (HRP) conjugated MAb 8F10, specific to mammalian sTnI, was used as the detection antibody. The assay conditions that were optimized include: the dilutions of the capture antibody and the detection antibody, the selection of the antibody buffer, the incubation time for antigen-antibody binding, and the dilutions of the adulterated meat and feed samples. The optimized assay achieved a detection limit of 0.05% (w/w) for raw, 0.50% (w/w) for cooked and 1.00% (w/w) for autoclaved beef in turkey (P < 0.05); 0.50% (w/w) for pork in chicken mixtures (raw, cooked and autoclaved) (P < 0.05); and 0.50% (w/w) for bovine meat meal in soy-based feed mixtures (P < 0.05). The fat content (0 − 30%, w/w) of the meat samples did not significantly affect the assay signals (P > 0.05). As the temperature and time of the heat treatment of the meat samples increased, the reactivity of this assay decreased slightly. However, the assay was still adequate to analyze samples subjected to the most severe heat treatment (132 degree Celsius/2.0 bar, 120 min). This MAb-based sandwich ELISA is the first assay suitable for rapid, sensitive and reliable detection of undeclared mammalian proteins in meat and feed products, regardless of the extent of heat processing.