The third-generation biofuels include biohydrogen (bioH2) production using microalgae is a green source of energy. There are several factors which inhibit the production of bioH2 such as oxygen (O2) production and a sufficient glucose concentration. A sufficient glucose concentration is vital for fermentative bioH2 yield; however, an insufficient glucose concentration may lead to halt the process. Whereas the production of O2 during the biophotolytic process is the primary inhibitor of bioH2 yield using algal biomass. It can be overcome using the co-culturing of algae and aerobic bacteria. For this purpose, activated sludge from a wastewater treatment plant was served as a source of bacteria in order to regulate O2 during co-culturing with Chlorella vulgaris (CCALA 256). It was observed that the co-culture with 1:1 v/v (algae : activated sludge) produced the highest amount of 1,177 mL/L of bioH2 with a minimum O2 content of 82 mL/L in six days of incubation as compared to the previous studies in which pure bacterial cells were used. The rest of the gas composition was mainly carbon dioxide (CO2) 34% and Nitrogen (N2) 15%, with no methane (CH4) production. Furthermore, a continuous supplementation of 1.5 g/L of glucose was also observed to be the enough to produce 1,084 mL/L of bioH2. It was also observed that anaerobic conditions, in result of co-culturing, may lead to trigger the hydrogenase activity in living algal cells which enhances the biophotolytic hydrogen production along with photofermentative hydrogen production. The substantial improvement in bioH2 production indicates the algal-activated sludge symbiosis as an alternative to pure algal-bacterial co-culture, which is source of green energy generation and waste energy utilizing process.