The type and concentration of inorganic carbon and nitrogen sources were manipulated to improve cell growth and lutein productivity of Desmodesmus sp. F51. Using nitrate as nitrogen source, the better cell growth and lutein accumulation were obtained under 2.5% CO2 supply when compared to the addition of NaHCO3 or Na2CO3. To solve the pH variation problem of ammonium consumption, the strategy of using dual carbon sources (NaHCO3 and CO2) was explored. A lower bicarbonate-C: ammonium-N ratio led to a lower culture pH as well as lower lutein productivity, but significantly enhanced the auto-flocculation efficiency of the microalgal cells. The highest biomass productivity (939 mg/L/d) and lutein productivity (5.22 mg/L/d) were obtained when the bicarbonate-C/ammonium-N ratio and ammonium-N concentration were 1: 1 and 150 mg/L, respectively. The lutein productivity of 5.22 mg/L/d is the highest value ever reported in the literature using batch phototrophic cultivation.