Cardiovascular disease is one of the biggest killing diseases in the world especially in Indonesia. The percentage risk of people stricken by this disease is so high in community and even causing death. This risk can actually be prevented with dietary addition of consuming unsaturated fatty acid that id often found in fish oil. However, the availability and quality of the fish in Indonesia's ocean is such that nowadays pollution has become the biggest threat. With the problem above, the alternative sources of omega 3 and omega 6 are needed whereas the source must be able to produce lipids in large number without taking up a large amount of land and also economic resources. There are several kinds of microorganisms that can be used as an alternative source of fatty acid. In this research, Rhizopus oligosporus is used as fungi microorganism. Mold is used in this research, because it has a lot of advantages such as ease to handle, easily grown in low pH and able to degradate more complex carbon sources. In this research, the fermentation is processed with submerged fermentation with temperature shift strategy with variation of glucose concentration and agitation rate. The results showed that the unsaturated fatty acid content obtained in the optimum conditions both from the glucose concentration of 100 g / L and agitation rate of 120 rpm are 41.77% in MUFA and 13.41% in PUFA and the highest highest oleic and linoleic acids were also obtained at 41.06% and 13.41%.