The culture medium requirement for invertase production by Pichia sp. was optimized and identified by initial screening method of Plackett–Burman. Furthermore, optimum concentrations of medium components, which were selected by in initial screening by Plackett–Burman, were determined by the Box-Behnken and its representative three-factor response-surface method. The regression models showed significantly high R² values of 97% for invertase activities, indicating that they are appropriate for predicting relationships between yeast extract, peptone and sucrose concentration with invertase production. According to the model the optimal concentrations of sucrose, yeast extract and peptone were 4, 5 and 4 g/ml, respectively. These predicted conditions were verified by validation experiments. In the optimized medium Pichia sp. produced invertase with activity of 38.71 U/ml, which is 4 times higher than that produced in original medium. Thus, this statistical approach enabled rapid identification and integration of key medium parameters for Pichia sp. BCCS M1, resulted the high invertase production.