Experiments of submerged cultivation of Monascus anka were carried out according to the sequential pseudo-uniform design (SPUD) method. Adopting the inputs including cultivation temperature and dissolved oxygen and the outputs of the model such as glucoamylase activity and concentration of extracellular red pigments, a second-order response model was successfully identified from the eleven experiments designed by the initial uniform design (UD) method followed by the SPUD method. The identified model achieved a 2.71% average modeling error for the glucoamylase activity but 12.44% error for the concentration of extracellular red pigments. Considering the resource for the experiments, the optimized operating conditions were determined based on the available experimental data to achieve the maximal overall desirability objective function of glucoamylase activity and concentration of extracellular red pigments of the product. The experimental results shown in this work successfully demonstrated the applicability of the SPUD method for locating minimal experiments that will effectively guide the experimenter toward optimized operating conditions of a new process.