Cardiovascular disease is the second biggest killer in Indonesia. The percentage risk of stricken by this disease is so high in community. Which are actually this disease can be prevent with dietary consumes of omega 3 and omega 6 such as AA and EPA. Omega 3 and omega 6 is often found in fish oil. However, the fish availability and the quality of Indonesia's ocean that tainted with heavy metals can be dangerous and also become the biggest problems of production omega 3 and omega 6 from fish. With the problem above the alternative sources of omega 3 and omega 6 is needed. Whereas the source must be able to produce oil in large number without taking a large amount of land and also economic. In this research, the fermentation of Aspergillus oryzae process with three-stage fermentation strategy with variation of time culture. This fermentation method divided into three stage where the first stage focused in cell propagation and second stage focused in lipid biosynthesis and the last stage for production of AA and EPA. In each stage the medium was design to be specific with the purpose. The time culture variation run in second and third stage. The most optimal cultivation time that run in second stage was 2 days with 17.2% lipid productivity or equal to 0.1341 g, on the other hand the most optimal cultivation time that run in third stage was 1 days with 54.51% Unsaturated fatty acid and 36.73% saturated fatty acid. The composition of unsaturated fatty acids is 29.1% oleic acid, 25.4% linoleic acid, 0.01% AA and 0.01% EPA.