By-product of tuna fish processing industry has the potential to be developed into fish oil rich in omega. Fish by-products are the main natural source of omega-3 polyunsaturated fatty acids, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) with a great importance in food industry and pharmacy. The purpose of this research is to see the effect of fish oil extracting method from the head of tuna fish as by-product of tuna fish processing industry to the physicochemical properties and the profile of fatty acid fish oil. There are several methods of extraction to produce of fish oil. This research was run in triplicate with a completely randomized design (CRD) with three treatments: pre-cooked wet rendering, acid silage, and solvent extraction. It can be concluded that the extraction method has an effect on physicochemical properties and fatty acid profile of fish oil. The wet rendering extraction method is the most effective and most potential extraction method to be applied because it produces the highest yield (12.80%) compared to the silage process (6.16%) and solvent extraction method (8.49%). PUFA produced from wet rendering was 44.34%, statistically not different with solvent extraction method (44.49%), but it higher than silage process method (32.77%).