The design of an optimum and cost-efficient fermentation conditions for production of cold-active cellulase by Penicillium cordubense D28 was attempted by using response surface methodology (RSM). Based on the Plackett–Burman design, temperature, seed age and inoculum size were selected as the most critical condition. Subsequently, they were investigated by the Box-Behnken design. Results showed that the maximum cold-active cellulase activity of 110.4U/mL was predicted when the coded values of temperature, seed age and inoculum size were -0.495, 0.455, and 0.253, respectively. The results were further verified by triplicate experiments. The batch reactors were operated under an optimized condition of the respective temperature, seed age and inoculum size of 17°C, 29h and 12.5%. The maximum cold-active cellulase activity of 219.9 U/mL was obtained based on the optimized fermentation conditions with further verified the practicability of this optimum strategy.