Since the 1940s, per- and polyfluoroalkyl substances (PFAS) have been widely produced and used in various applications due to their unique properties. Consequently, the principal exposure routes of PFAS have been broadly studied, leading to the conclusion that dietary exposure (more specifically, the consumption of fish and seafood) was one of their main contributors. Thus, developing an analytical method that determines the level of PFAS in fish and seafood has become a relevant subject. In this work, a previous analytical method has been optimized to determine 12 PFAS in fish muscle from salmon, tuna, cod, hake, sardine, anchovy, and sole, as well as in seven different seafood species (i.e., cuttlefish, octopus, squid, shrimp, Norway lobster, prawn, and mussel) by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Subsequently, the PFAS profile of the different species was studied to determine if it was consistent with that previously reviewed in the literature and to know the most relevant contribution of PFAS for each species. Finally, human exposure to PFAS through their consumption was estimated by the daily intake for seven different age/gender groups. PFAS were obtained from 0.014 to 0.818 ng g−1 wet weight in fish samples. Sardines, anchovies, and soles presented the highest PFAS levels. However, cod samples also showed some PFAS traces. Regarding seafood, PFAS levels range from 0.03 to 36.7 ng g−1 dry weight for the studied species. A higher concentration of PFAS has been found in the cephalopods' spleens and the crustaceans' heads. PFOS and PFBS were the predominant compounds in each seafood species, respectively. On the other hand, in the case of mussels, which are the less polluted species of the study, contamination by longer-chained PFAS was also observed. Finally, the total intake of PFAS due to fish and shellfish consumption for the Spanish adult population was estimated at 17.82 ng day−1. Nevertheless, none of the analyzed samples exceeded the European Food Safety Authority (EFSA) risk value for the supervised PFAS in any age/gender group reviewed.