Pakistan utilizes huge amount of budget to import petroleum products from other countries because very limited resources of fossil fuels are available in country. However combustion of fossil fuel increases concentration level of greenhouse gases, those are destroying environment. There is a need of some alternate and cheaper resources of energy to overcome the deficiency of fuels. There are number of challenges for the marketable applications of the advanced technologies of fuel production which includes pretreatment methods and enzymes production. For the microbiologist and ecologist, termite gut represents an outstanding representative of highly structured micro environments and therefore also called as mini biorefineries. Because the decomposition of lignocelluloses is proficiently perform by these soil insects. A study was conducted to develop a method for the production of bioethanol from agricultural waste materials which includes corn stover, cotton stalk and rice straw by using cellulolytic bacteria from termite gut. Bacteria were isolated from termite gut and identified by 16S rRNA gene sequencing. The isolates were screened for CMCase and xylanase activity. Three isolates were selected among which two were bacillus sp. and the one was cellulomonas sp. Their avcilase CMCase and xylanase activities were studied on selected temperature and pH. It was found that all of the bacterial isolates have shown higher avicelase activity at 40 oC, CMCase and xylanase activity at 50 oC. Isolate 9x, 10 and 31 have shown higher avicelase activity at 7 pH, whereas CMCase activity was found to be optimum at pH 8. In case of xylanase activity isolate 9x and 10 showed higher activity at pH 6 and isolate 31 at pH 7. Biological and chemical pretreatments were compared for corn stover, cotton stalk and rice straw. Efficiency of enzymes of bacterial isolates for saccharification of agricultural substrates was assessed. It was observed that xylanase (isolate 9x) 1 2 released more sugar amount from corn stover as compared to other substrates. Direct saccharification and fermentation of corn stover and rice straw was carried out using all three bacterial isolates without using pretreatment methods. Ethanol concentration was analyzed by using High performance Liquid chromatography. It was observed that isolate 31 has produced higher amount of ethanol by using corn stover. Therefore it was found that these bacterial enzymes have the potential to hydrolyze not only pure substrates but can also degrade agricultural substrates. Bacterial isolates obtained from termite gut have potential to can convert different agricultural substrates into valuable bioproducts including various alcohols (ethanol) which may have new innovative source for producers.