Bacillus cereus ZH14 was previously found to produce a new type of antiviral ribonuclease, which was secreted into medium and active against tobacco mosaic virus. In order to enhance the ribonuclease production, in this study the optimization of culture conditions using response surface methodology was done. The fermentation variables including culture temperature, initial pH, inoculum size, sucrose, yeast extract, MgSO(4).7H(2)O, and KNO(3) were considered for selection of significant ones by using the Plackett-Burman design, and four significant variables (sucrose, yeast extract, MgSO(4).7H(2)O, and KNO(3)) were further optimized by a 2(4) factorial central composite design. The optimal combination of the medium constituents for maximum ribonuclease production was determined as 8.50 g/l sucrose, 9.30 g/l yeast extract, 2.00 g/l MgSO(4).7H(2)O, and 0.62 g/l KNO(3). The enzyme activity was increased by 60%. This study will be helpful to the future commercial development of the new bacteria-based antiviral ribonuclease fermentation process.