Today the main source of unsaturated fatty acids especially omega-3 is still from marine fish oil. In this research, Rhizopus oligosporus will be used to produce PUFA (polyunsaturated fatty acids). This research used submerged fermentation method and temperature shift strategy to optimize the production of PUFA. In this result, batch culture of R.oligosporus for PUFA production at various temperatures (22, 26, 32, and 37°C). Result showed that R.oligosporus has different optimum temperature for growth and PUFA production, 26°C and 22°C respectively. Based on the result temperature-shift strategy was applied, in which the culture temperature was controlled at 26°C at the first 5 days, and then switched to 22°C for two days. By applying such a temperature-shift strategy, the dry cell weight decreased from 0,55 g to 0,47 g and the concentration of PUFA increased from 19,8% to 22,5%.