As fish and other seafoods are exceptionally fragile, to maintain and monitor their safety and quality, it is important not only to employ processing technologies but also to develop methods, that are rapid, sensitive and precise. With rapid technological developments over the last two decades, fluorescence spectroscopy has shown its potential for rapid and noninvasive quality analysis of fish and fish products, which is cost-effective and can even achieve on-line detection. This chapter gives an overview of the use of fluorescence spectroscopy especially highlighting the potential of excitation–emission matrix (EEM) or fluorescence fingerprints for determining changes in fishery products and their quality and authenticity during technological process and storage. Typical intrinsic fluorophores in seafood are aromatic amino acids, adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH), vitamin A, riboflavin, and oxidation products. Out of different data acquisition modes of fluorescence spectroscopy, EEM is advantageous for the quantification of fluorophores present in different areas, and visualizing all fluorophores that exist in the sample even in the frozen state. Finally, we have also introduced multidimensional fluorescence imaging, a combination of fluorescence EEM and imaging techniques for visualizing seafood quality.