This study was aimed to optimize the process variables for improved production of biomass protein using from banana fruit peel by the use of response surface methodology. A five-level-four factors central composite rotatable design was applied to elucidate the influence of process variables viz. temperature (20-40 °C), pH (4-8), substrate concentration (5-25%), and fermentation period (1-5 days) on biomass and protein content. The second-order polynomial models were established, which effectively explicated the variation in experimental data and significantly epitomized the appreciable correlation between independent variables and responses. After numerical optimization, the predicted optimum conditions (temperature of 31.02 °C, pH of 6.19, substrate concentration of 19.92%, and the fermentation period of 4 days) were obtained with biomass of 24.69 g/L and protein of 61.23%, which were verified through confirmatory experiments.