Abstract: The fermentation conditions for killer toxin production from Cyberlindnera mrakii WM1 preserved in the laboratory of marine yeast resource pool were optimized. The diameter of inhibition zone ( mm) was used as evaluation index and quantified against Canidia albicans YTS-03. Firstly, chose inoculum concentration, p H value, fermentation temperature and time as independent variables, based on these single factor tests, response surface methodology with the four variables was employed to optimize the fermentation conditions for killer toxin production. The optimum fermentation conditions of Cyberlindnera mrakii WM1 production for the growth and metabolism of killer toxin were inoculation of 8%, p H of 4.0, fermentation temperature of21 ℃, fermentation time of 2.5 days. Under these optimal conditions, the WM1 killer yeast fermentation broth reached its maximum capacity of 21.00 mm, than before optimization ( 16.5 mm) to improve 27.3%.The number of viable cells also reached2.58 × 10⁸CFU/m L, compared with the previous optimization ( 1.9 × 10⁸CFU/m L) improved 35.8%. At the same time, the experimental results obtained under the optimal fermentation conditions were in agreement with the predicted values, indicated the established regression model was feasible.