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Response Surface Methodology (RSM) is a powerful and efficient mathematical approach widely applied in the optimization of cultivation process. Cellulase enzyme production by Trichoderma reesei RutC30 using agricultural waste rice straw and banana fiber as carbon source were investigated. In this work, sequential optimization strategy based statistical design was employed to enhance the production of cellulase enzyme through submerged cultivation. A fractional factorial design (26-2) was applied to elucidate the process parameters that significantly affect cellulase production. Temperature, Substrate concentration, Inducer concentration, pH, inoculum age and agitation speed were identified as important process parameters effecting cellulase enzyme synthesis. The concentration of lignocelluloses and lactose (inducer) in the cultivation medium were found to be most significant factors. The steepest ascent method was used to locate the optimal domain and a Central Composite Design (CCD) was used to estimate the quadratic response surface from which the factor levels for maximum production of cellulase were determined.