Euglena is one of the alternative natural resources for medicine, food, and energy, and it is important to develop its metabolic contents to fulfill human demands. Improvement of metabolic content in Euglena was conducted in several ways, such as by adding the phytohormone. Brassinolide is one of the phytohormones and is well-known for its ability to stimulate and protect the plant from stressful environments. The application of brassinolide is still lacking. In addition, previous studies have never applied this phytohormone to Euglena sp. cultures. This research aimed to analyze the effect of brassinolide on the growth, metabolic content and wax fermentation in Euglena sp. The growth rate was measured during cultivation, and the metabolic content was analyzed at the late exponential phase before entering the fermentation process. Gas Chromatography-Mass Spectrometry (GC-MS) was carried out to reveal the wax ester content after the fermentation process. The result showed that brassinolide significantly increased the growth rate and metabolic content at lower concentrations, while high concentration tends to inhibit the effect. The high metabolite content, including carbohydrate, lipid, protein, and paramylon, was 0.47 ± 0.02 g/L, 0.20 ± 0.01 g/L, 15.91 ± 1.21 × 10^-3 g/L, and 145 ± 0.10 × 10^-3 g/L, respectively. Interestingly, wax esters at lower brassinolide concentrations showed contrasting results compared to the control treatment. These findings provide information about the effect of brassinolide in Euglena sp., and advanced research is needed to reveal the mechanism of brassinolide in Euglena sp.