To take advantage of protein resource from cold-pressed peanut meal, and to reduce the impact of residual oil on protein deep processing, anhydrous ethanol was chosen as defatting solvent after comprehensive consideration of solvent safety and the influences of anhydrous ethanol on protein loss rate and defatting rate. The influence rules of defatting temperature(X1), defatting time(X2) and solid-liquid ratio(X3) were investigated by quadratic rotatable central composite response surface methodology based on single factor experiment, and a second order polynomial nonlinear regression equation and numerical model was established. Optimum parameters were determined as defatting temperature 59.5 ℃, defatting time 108 min and solid-liquid ratio 1∶19. Under the optimal condition, defatting rate was 92.56%, which was close to the model predicted value 93.07% with the deviation of 0.55%. The results indicated that the combination of single factor experiment and quadratic rotatable central composite response surface methodology can be used in optimization of defatting process of cold-pressed peanut meal.