The culture conditions and media composition for the heterotrophic culture of an axenic strain of Ettlia sp. YC001 were firstly optimized using the Plackett-Burman design (PBD) and response surface methodology (RSM). The strain successfully showed higher productivity in the basal media without any light illumination at 32.2 to 33.3 °C. The PBD results showed that the most effective components for biomass productivity of Ettlia sp. were fructose and yeast extract for sources of C and N, respectively. The RSM results showed an optimal level of 72.2 g/L for fructose and 21.5 g/L for yeast extract, resulting in 46.1 g/L biomass with a lipid content of 13.8% over a course of 9 days. Using a 5 L scaled-up fermentation system for 6 days, the production of biomass and lipids was 7.21 g/L/day and 1.18 g/L/day, respectively. Consequently, heterotrophic cultivation of Ettlia sp. YC001 provided much higher production of biomass and lipids than those of autotrophic cultivation. As further research, the use of substitute substrates instead of fructose and yeast extract should be developed to reduce production costs.