The present study was carried out with the production of ethanol by Aspergillus niger, Rhizopus oryzae, Saccharomyces cerevisiae and baker's yeast using banana pseudostem as a substrate. Ethanol production was optimized by different pH and temperature under fermentation process. Maximum amount of ethanol concentration of 3.82±0.4 g/100 ml was produced at pH 5 and temperature 30°C. Maximum amount of ethanol produced by Aspergillus niger, Rhizopus oryzae and Saccharomyces cerevisiae inoculated media was analyzed by HPLC. From the results it is evident that the aminoacid present in samples were Serine, Histidine, Lysine, Aspartic acid, Glutamic acid, and Tryptophan. Since these compounds were found to be present in the sample, it might to be responsible for the ethanol production. It was observed that pretreated banana pseudostem can be economically utilized as a cheaper substrate for ethanol production
