Microalgae, such as Euglena sp., are microorganisms that can be utilized as biofuel sources. Before mass cultivation, Euglena sp. must adapt through semi-mass cultivation to tolerate various conditions, including environmental changes and contaminants. Nitrogen starvation has been shown to increase lipid production in Euglena sp.; however, the effects of culturing with ((NH4)2SO4) concentrations of 500 mg/L and 250 mg/L have not yet been explored. This research aimed to determine the effects of ((NH4)2SO4) starvation on growth rate and biomass, lipid content, and fatty acid profile of Euglena sp. during semi-mass cultivation (50 L). In this study, nitrogen starvation increased the total unsaturated fatty acids, lipid production, and productivity. However, it reduced cell growth, biomass production, and productivity. The highest lipid productivity was observed at 500 mg/L ((NH4)2SO4), reaching 0.011±0.0014 mg/mL and 0.217±0.014 mg/mL for lipid production on day 15. Meanwhile, the highest biomass production occurred at 1000 mg/L ((NH4)2SO4), reaching 0.156±0.010 mg/mL on day 15 and a biomass productivity of 0.0067 mg/mL. Methyl palmitoleate (C16:1) was the dominant FAME in all treatments, reaching 18.38% at 500 mg/L ((NH4)2SO4). Thus, it can be concluded that the 500 mg/L ((NH4)2SO4) treatment is more efficient in increasing FAME, lipid production, and productivity for biofuel production.