Abstract: Using mixture design to optimize formula of mixed bacteria, we studied the effects of 4 strains of bacteria on OD₆₀₀, ammonia and nitrite removal rate, and established a regression model. Through the equation analysis and optimization of Design Expert 8.0, the response values satisfying all expectations were optimized, and the most excellent combinations of Bacillus sp. BD6, Bacillus sp. BZ5 and Vibrio alginolyticus VZ5 were 5.2%, 22% and 72.8%, respectively. The result of verification experiment on the formulation was consistent with the prediction. Ammonia and nitrite removal rate by the mixed bacteria was higher at 24^th hour, reaching 98.37% and 93.81% at 72^th hour, respectively. Mixture design and analysis software can optimize the formulation of mixed bacteria for several responses and obtain the best formulation.