This study employs the Box–Behnken Design (BBD) of Response Surface Methodology to optimize key variables influencing biomass production in Chlorella vulgaris. A second-order polynomial model demonstrated strong predictive capability for biomass yield (P < 0.0001). Under optimized conditions—pH 8.07, temperature 39.5 °C, sodium bicarbonate 0.007 mg/mL, and urea 0.005 mg/mL—the model predicted a biomass yield of 11.165 mg/mL. Model performance showed residuals greater than 2.34%, with R² of 98.32 and adjusted R² of 90.83, indicating a reliable fit. Chlorella vulgaris cultivated in modified artificial sea nutrient medium (III) enriched with CO₂ produced lipids that were extracted using a Soxhlet apparatus. GC–MS analysis identified 35 lipid compounds, while transesterified samples revealed 30 biodiesel-related compounds along with 20 compounds in crude glycerol. The transesterification process yielded 86.115% biodiesel compounds and 92.015% crude glycerol, outperforming the Soxhlet extraction method, which produced 53.56% biodiesel compounds. These findings highlight the effectiveness of BBD optimization and demonstrate the potential of Chlorella vulgaris biomass for biodiesel production. © 2022 Elsevier B.V., All rights reserved.