<p>The study employed a central composite design (CCD) response surface method (RSM) to optimize initial CO₂ levels, nitrate concentrations, and ZnO nanoparticle concentration in the culture media for enhancing biomass, lipid, and carbohydrate yield in <i>Chlorella pyrenoidosa</i>. The predicted second-order quadratic model for response variables was found to be significant (<i>p</i> &lt; 0.01), and the analysis of variance (ANOVA) showed a high coefficient of determination with <i>R</i>² being 0.99 for biomass, 0.98 for lipid, and 0.99 for carbohydrate. The maximum biomass, lipid, and carbohydrate yield of 1.5 ± 0.06&#xa0;g L⁻¹, 43.6 ± 1.9%, and 22.8 ± 3.1%, respectively, were obtained by RSM optimized culture medium containing an initial CO₂ concentration of 1.31&#xa0;g L⁻¹, a nanoparticle dose of 50&#xa0;mg L⁻¹, and a nutrient concentration of 150&#xa0;mg L⁻¹. The lipid and carbohydrate yield in the optimized conditions are 60% and 85% higher than in microalgae grown in commercially available BG11 media. This research demonstrates that the targeted optimization of CO₂, nitrate, and ZnO nanoparticle concentrations can significantly enhance the biomass, lipid, and carbohydrate yield in <i>C. pyrenoidosa</i>. These findings underscore the viability of using <i>C. pyrenoidosa</i> as a feedstock for biofuel production, particularly in biodiesel and bioethanol applications.</p> Graphical abstract <p></p>