Glucose added as an organic carbon source has been shown to significantly influence the growth and biochemical composition of Chlorella pyrenoidosa under mixotrophic conditions. Variations in glucose concentrations led to changes in cell metabolic patterns, which were reflected in growth performance and the accumulation of key biomolecules. Glucose supplementation at a concentration of 15 g/L showed the most optimal response, with a more than threefold increase in biomass compared to the control without glucose. This condition also accelerated the specific growth rate and decreased the cell doubling time by approximately 30%, indicating that cells were able to utilize energy from photosynthesis and glucose respiration simultaneously to power the division process. The higher availability of glucose directed cell metabolism toward the formation of storage compounds. This was indicated by a decrease in protein levels to approximately 35% of the initial value in the control, indicating a diversion of metabolic resources from protein synthesis to the production of storage compounds. Conversely, lipid levels increased almost twofold at higher glucose concentrations, reflecting the microalgae's adaptive response of storing excess carbon in the form of fatty acids. Meanwhile, total sugar levels showed an increase pattern at low glucose concentrations, before decreasing again at high concentrations in line with the metabolic shift towards lipid formation. Overall, the results of this study confirm that the addition of glucose can increase growth efficiency and shift the metabolic direction of Chlorella pyrenoidosa towards the accumulation of high-value biomolecules. This provides a strong scientific basis for the use of glucose as an organic carbon source to optimize the production of biomass and important metabolites in mixotrophic microalgae culture systems.