Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.