Abstract: A considerable attention is given to biofuels by scientists at the global level; this type of energy is attracting more and more attention, due to various factors such as the need to increase energy security and overcoming the problem of energy emissions and greenhouse gas causing global warming and many other problems. The purpose of this present study is to demonstrate the potentialities of the green alga CLADOPHORA Sp collected from Ouargla region in southern Algeria, as a source of energy of third generation, after the characterization of the algal biomass, the results show that this species is rich in carbohydrate, after the extraction, the yield acquired is very interesting, it is of the order for 75.12% / DW of total sugar, The third generation bioethanol production process is carried out in four main stages: pretreatment, hydrolysis, fermentation and separation. The alcoholic fermentation in anaerobic medium gave a significant yield, which is of the order of 30.85% (v/v) of bioethanol; the rate of glucose converted in bioethanol is 68.11%. The characterization of bioethanol as well as the mixture of bioethanol with unleaded gasoline prepares to 10% (SP95E10) to give results in accordance with the international standards in force. In effect, based on the results above, this present work allows us to conclude that, the green alga CLADOPHORA Sp accumulates high carbohydrate content , it has significant capacity for biofuel production and constitutes a choice delicate for operate as raw material for the production of the third generation energy, that is clean, sustainable and renewable.