Abstract: In liquid culture, 18 kinds of carbon and 18 kinds of nitrogen resources were selected for best nutritional agents. The effects of nutrition on the volatiles of Bacillus weihenstephanensis strain MC67 with highest nematicidal activity to Bursaphelenchus xylophilus were determined by single factor analysis. The best carbon and nitrogen resources were maltose and yeast extract, respectively. Plackett-Burman design and response surface analysis were used to optimize the medium constituents and fermentation conditions of B. weihenstephanensis. The important factors were determined as follows: maltose, yeast extract, pH value and temperature. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical factors, and to find out the optimal concentration levels and fermentation conditions. By solving the quadratic regression model equation using appropriate statistic methods, the optimal level of the variables were determined as: maltose 10.6 g/L, yeast extract 0.66 g/L, culture temperature 29.6℃ and pH value 6.6. In the most suitable condition, the nematicidal activity of MC67 could reach to 98.2%,which validated the theoretical value 99.1%.