Fish and seafood is tasty, nutritious and healthy, increasing its demand on a global level. In this regard, the fish processing industry and aquaculture are becoming the most important sectors of food production. The trend towards the production of ready-to-eat high-quality products requires more processing, where large amounts of by-products are created, and instead of throwing them away, the use of by-products is becoming an important industry. By-products produced in the fish processing industry contain many high-value components such as polyunsaturated fatty acids, bioactive peptides, collagen, enzymes, minerals, vitamins, etc. Bluefin tuna (Thunnus thynnus), sardine (Sardina pilchardus), sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) are important economic species of Croatian fish industry, and their processing produces by-products that are potentially high-quality secondary raw materials for the production of fish oil. The aim of this work was to produce and characterize crude oils from by-products of the mentioned species and to purify them without losing important components. The highest proportion of lipid and the highest yield of oil were recorded for tuna by-products. In crude oils and during individual stages of refining, oil quality parameters and fatty acid composition were determined. All measured parameters of crude and refined oils were within the standard values for high-quality fish oils. Chemical refining through four stages proved to be effective in reducing impurities in oils, which resulted in reduced values of free fatty acids, peroxide number, anisidine value, malondialdehyde, and improved oxidation stability of the oil. After refining, the oils retained their content of highly unsaturated fatty acids and a favorable n-6/n-3 ratio, and the composition of volatile compounds responsible for the sensory profile of the oil improved. The use of alternative raw materials for the production of fish oil contributes to the sustainability of the fishing sector. The production of oil from the by-products of the fishing industry can offer a solution that will improve waste management and the ecological aspect of fish processing and add value to the waste.