Microalgae use solar energy, nutrients and carbon dioxide to produce protein, carbohydrates, fats and some valuable compounds, such as carotenoids. These microorganisms are used in various fields such as food and feed, health and biofuels production. The biological refinery is referred to a set of industrial processes in which alga biomass is converted to biochemical, fuel, food, pharmaceutical and valuable products. In this research, the potential of brown microalgae Isochrysis aff. galbana in a biorefinery mode was investigated to produce significant percentage of fat, pigment fucoxanthin and docosahexaenoic acid. By examining the rich culture media Conway and F/2, Conway medium was chosen as the main cultivation medium due to production of 16.15 mg fucoxanthin /g dry weight of microalgae. By investigating the effect of nitrate concentration as a source of nitrogen (low, high and supplemental nitrate concentrations), it was found that the lowest amount of fucoxanthin produced was in low nitrogen concentration with 7.79 mg/g dry weight and the highest amount of fucoxanthin was related to supplemental and high nitrogen concentration with 20.09 and 18.88 mg/g respectively that indicating the significant effect of nitrate content of cultivation medium on the production of fucoxanthin. To study the light parameter, various optical conditions of red LED, blue LED and mixed LED (red and blue with a ratio of 50:50) were investigated. The lowest amount of fucoxanthin produced from microalgae in red light with 14.77 mg/g dry weight and the highest amount of fucoxanthin was obtained in blue and mixed with 19.08 and 18.30 mg/g dry weight, respectively. The amount of lipid produced from microalgae in blue and mixed light was 21.41% and 24.3% respectively, which are more than that produced in red light. A two stage culture method was developed to increase the amount of biomass in the first stage and increase the content of lipid in the second stage. Therefore, the Conway culture medium with high nitrate and mixed light conditions were selected for the first stage, with the goal of increasing lipids, fucoxanthin and biomass by microalgae and nitrogen deficiency culture medium was chosen for the second stage. The amount of fucoxanthin produced by microalgae in the first and second stages of two-stage cultivation was 20.62 and 17.72 mg/g, respectively, which was increased compared to normal culture (Conway culture medium and fluorescent white light). On the other hand, the amount of fucoxanthin was reduced in the second stage of the two-stage culture compared to the first stage. It can be concluded that under nitrogen-poor conditions, by reducing the photosynthetic pigments, such as chlorophyll a and b, enhancement of the carotenoids is due only to photoprotective carotenoids. In two-stage cultivation, the amount of produced lipid was 65 percent higher than conventional cultivation and the biomass productivity was approximately equal to normal culture and more than that of nitrogen deficiency culture. According to the results, it can be concluded that microalgae in a two-stage culture due to increased biomass productivity in the first stage results in higher lipid production with a yield of 0.251 g/l. Although two-step cultivation reduced the amount of docosahexaenoic acid from 12.2% to 9.82% compared to normal culture, the ratio of omega-3 to omega-6 still remained within the limits of nutritional standards. These results indicate that applying two-step cultivation increases the amount of saturated fatty acids to produce biodiesel, as well as the remaining biomass for aquaculture. Keywords: