The aim of proteomics is to simultaneously resolve all potential proteins expressed by a cell, tissue or organism in a specific physiological condition. It will allow scientists to build and test better hypotheses, with the ultimate goal to find better solutions to challenges in agricultural sciences, medicine and environmental management. Recent and very promising applications of proteomics have also been provided in the field of aquaculture, such as the search for antigenic proteins, the detection of differentially regulated proteins and the characterization of biologically active proteins primarily to investigate the physiology, development biology and impact of contaminants in aquatic organisms. Thus, a brief overview of present classical proteomics methodology (electrophoresis and mass spectrometry) is presented, and the proteome technique will be focused in the context of applications beneficial to the field of fish, shellfish and seafood/seafood product. In addition, the future potential application and challenge of proteomics in aquaculture will also be discussed.