Macroalgae with several species are an abundant, carbon-neutral renewable resource and rich in carbohydrates which make it suitable for biobutanol production. Recently, due to advantages of biobutanol as a liquid biofuel, it can be used as a substitute for gasoline and diesel. Many researches mentioned that, different Clostridium species are able to use fermentable sugars for production of biobutanol from different biomasses through ABE (Acetone- Butanol- Ethanol) fermentation process. In this study the (Sargassum sp.) was used as carbon source for biobutanol production. Thirty-three anaerobic mesophilic isolates were isolated on RCM medium from five soil cultivated with different crops. Only thirteen spore forming, mesophilic, anaerobic Clostridium isolates used for the fermentation process. 100 g/L from Sargassum sp were hydrolyzed by sulfuric acid (6 % (v/v)) followed by thermal pretreatment at 121°C for 20 minutes to produce total reducing sugars (24.151 ± 0.273 g/L), which were fermented by Clostridium isolates. The most promising isolate (HG1) which produce the highest butanol level, acetone and ethanol (3.743, 1.401 and 1.031 g/l) respectively was identified according to Butanol dehydrogenases (bdhA) gene analysis as Clostridium acetobutylicum. This paper emphasizes the importance of (Sargassum sp.) as a renewable feedstock for the biobutanol production.