The use of lignocellulosic material is the second-generation process of Bioethanol production and there is a sufficient supply of lignocellulosic materials in agricultural industries such as wheat bran, cotton ginning waste and sugarcane bagasse. The huge amount of waste generated from cotton ginning industry is a burning issue to dispose off, so in order to curb that issue, fermentative bioethanol production from cotton ginning waste using Aspergillus spp. is the best possible options to overcome it. It is also helpful in alleviating the global problem of greenhouse gases and decreasing the use of non-renewable energy with the generation of bio fuel using cotton ginning waste. The key stages involved in Bioethanol production from lignocellulosic biomass consist of pretreatment of biomass, saccharification, fermentation, and Ethanol Production. In current research work, the combination of three different methods for pretreatment of cotton ginning wastes: acid, alkaline and acid-alkaline were evaluated for the conversion of cellulose and getting higher ethanol yield. After Pre-treatment, sugar i.e., cellulose was further exposed for enzymatic hydrolysis by the cellulase enzyme produced from Aspergillus Spp. using Solid State Fermentation (SSF). The saccharified substrate was then utilized as C source for the fermentation and production of Bioethanol. The results of pre-treatment succeeded that acid pre-treated, alkaline pre-treated and acid-alkaline pre-treated substrates Cotton ginning wastes (CGW) released 172.8 mu g/ml, 256 mu g/ml and 140.8 mu g/ml of sugar, respectively. The sugar released were then fermented with Saccharomyces cerevisiae using SHF method (Separate Hydrolysis and Fermentation). The amount of ethanol produced from Acid pre-treated, Alkaline pre-treated and Acid-Alkaline pre-treated Cotton ginning wastes (CGW) was 48 mg/ml, 48 mg/ml and 60 mg/ml, respectively. Thus, based on current experimental work it can be stated that the lignocellulosic biomass Cotton Ginning Wastes (CGW) can be converted into biofuel (bioethanol) using Separate Hydrolysis and fermentation process which is the best alternative source of fuel.