The marine microalgae Nannochloropsis oculata is a promising source of biofuel because of its high lipid content. For achieving high productivity of oil from microalgae, a high cell concentration before harvesting is beneficial. The present study investigated fed-batch cultures of N. oculata fed with vitamins and nutrient solutions and found that the biomass yield of N. oculata in the fed-batch culture was 1.25 times higher than that in batch culture. Fed-batch cultivation, especially at high illumination, decreased the inhibitory effect of high carbon dioxide (CO 2 ) concentration on the microalgal growth. The specific growth rate was directly proportional to the light intensity in the CO 2 environment. A light intensity of 40,000 Lux was able to achieve high specific growth rates in fed-batch cultivation at a CO 2 volume fraction of 2%–15%. The tolerance of N. oculata to CO 2 was enhanced by the daily feeding of nutrients in the fed-batch cultivation. At 2% CO 2 , a final cell density of about OD 682 = 11.4 was achieved in the fed-batch culture in 30 days. Furthermore, a cell density of 14.4 g L −1 was obtained by outdoor fed-batch cultivation in 27 days.