Bio-butanol is more useful than bio-methanol and bio-ethanol due to higher energy density. In current study, we carried out bio-butanol content, yield, carbohydrate, protein content, biomass concentration, biomass productivity, and specific growth curve of Chlorella zofingiensis cultivated in various concentrations (25, 50, 75 and 100%) of municipal wastewater and Indole-3-acetic acid (IAA) via hydrogen peroxide (H2O2) pre-treatment. Also, we investigated the activities of photosynthetic pigments and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) on C. zofingiensis under stress conditions. To our results, carbohydrate percentage of C. zofingiensis increased from 23.9 to 34.2% at 25% wastewater and Indole-3-acetic acid 80 mu M after pre-treatment of hydrogen peroxide (4%). At these conditions, bio-butanol content was 0.084 and it corresponds to an increase of 35%. The combined use of IAA, wastewater and hydrogen peroxide can be new challenge for production of high amounts of bio-butanol after membrane filtration technology develops for the removal of contaminants such as bacteria and fungi.