In order to produce microalgal lipids that can be transformed to biodiesel fuel, effects of concentration of CO₂ aeration on the biomass production and lipid accumulation of Nannochloropsis oculata in a semicontinuous culture were investigated in this study. Lipid content of N. oculata cells at different growth phases was also explored. The results showed that the lipid accumulation from logarithmic phase to stationary phase of N. oculata NCTU-3 was significantly increased from 30.8% to 50.4%. In the microalgal cultures aerated with 2%, 5%, 10% and 15% CO₂, the maximal biomass and lipid productivity in the semicontinuous system were 0.480 and 0.142gL⁻¹ d⁻¹ with 2% CO₂ aeration, respectively. Even the N. oculata NCTU-3 cultured in the semicontinuous system aerated with 15% CO₂, the biomass and lipid productivity could reach to 0.372 and 0.084gL⁻¹ d⁻¹, respectively. In the comparison of productive efficiencies, the semicontinuous system was operated with two culture approaches over 12d. The biomass and lipid productivity of N. oculata NCTU-3 were 0.497 and 0.151gL⁻¹ d⁻¹ in one-day replacement (half broth was replaced each day), and were 0.296 and 0.121gL⁻¹ d⁻¹ in three-day replacement (three fifth broth was replaced every 3d), respectively. To optimize the condition for long-term biomass and lipid yield from N. oculata NCTU-3, this microalga was suggested to grow in the semicontinuous system aerated with 2% CO₂ and operated by one-day replacement.