Hydrogen peroxide was applied for promoting sporulation of Esteya vermicola and response surface methodology was used to optimize the effect of processing parameters on sporulation. Three variables were concentration (X-1), treatment time (X-2), and carbon-to-nitrogen ratio (X-3). The results indicated that X-1 and X-2 and the quadratic term of X-1 had significant effect on the sporulation, followed by the significant interaction effects between X-1 and X-2. The optimal conditions of promoting sporulation were as follows: hydrogen peroxide concentration 1.65 mM, treatment time 9.40 min, and carbon-to-nitrogen ratio 9: 1. Under these conditions, sporulation increased twelve times compared with control and this result was in agreement with model predictions.