Identification of fish species adulteration is important for consumer protection and the enforcement of food-labeling laws. A monoclonal antibody (MAb) generated against soluble muscle proteins from grouper (Epinephelus guaza) has been used in two indirect enzyme-linked immunosorbent assay (ELISA) formats (microtiter plates and immunostick tubes) for the rapid authentication of grouper fillets. The 3D12 MAb was produced with the use of the hybridoma technique and tested against several commonly consumed fish species by ELISA. The 3D12 MAb specifically reacted with grouper samples and could be useful for the discrimination of grouper among other, less-valued, fish species sold in the marketplace.