The aim of this study was to improve the protein content and yield of heterotrophic microalgal cultivation and establish a simple, economical, and efficient method for microalgal protein production using the novel green alga,Graesiella emersoniiWBG-1, which has not been previously reported for heterotrophic cultivation. Through batch heterotrophic cultivation of this alga, we observed that glucose was the optimal carbon source, while it could not use sucrose as a carbon source. Biomass production and protein content were significantly reduced when sodium acetate was used as the carbon source. Compared with nitrate, protein content increased by 93% when urea was used as the nitrogen source. Cultivation temperature had a significant impact on biomass production and protein content. The optimal conditions were glucose as the carbon source at an initial concentration of 10 g/L, urea as the nitrogen source at an initial concentration of 1.62 g/L, and a culture temperature of 35 °C. On the second day of batch cultivation, the highest protein content (66.14%) was achieved, which was significantly higher than that reported in heterotrophic cultures ofChlorellaand much higher than that reported for specially established technologies aimed at increasing the protein content, such as two-stage heterotrophic, heterotrophy–dilution–photoinduction, and mixotrophic processes. These results demonstrate the great potential of the heterotrophic cultivation ofG. emersoniiWBG-1 for protein production.