The banana peduncle was used as a substrate for the production of bioethanol using Trichoderma reesei MTCC 4876 and Kluveromyces marxianus MTCC 1389 by separate saccharification and fermentation. The banana peduncle was pre-treated by co-pretreatment method using different hydrolytic processes such as acidic and thermo-chemical methods as well as a combination of ultrasonication. Fermentation process conditions such as temperature, pH, agitation speed, inoculum size and substrate concentration were optimized using Response surface methodology (RSM) and Artificial neural network (ANN). Maximum bioethanol concentration of 25.39 g/l was obtained at the optimal conditions of temperature 41 °C, pH 4.5, agitation speed 170 rpm, inoculum size 3.6 %(v/v) and substrate concentration of 4% (w/v) using ANN method. The biomass growth, substrate utilization, and production formation kinetics were studied. The experimental biomass concentration of 15 g/l with a specific growth rate of k = 0.13 h−1, bioethanol concentration of 25.4 g/l and the substrate concentration of 5.44 g/l were obtained from the kinetic analysis. The produced bioethanol was analysed and confirmed using GC-FID, FT-IR and NMR analysis.