Plackett-Burman (PB) and Box-Behnken (BB) experimental designs were used to optimize fermentation variables for the biotransformation of glycyrrhizin (GL) to monoglucuronyl-glycyrrhetinic acid (MGGA). The PB design was first used to screen the important factors among the medium variables. The steepest ascent method was used to approach the optimum range for each of these factors. The BB design was finally used to analyze the response surfaces of the screened factors for further optimization. The optimized conditions for this system were 0.7 g/L MgSO4·7H2O, 1.19 g/L GL, and cultivation for six days. The biotransformation of GL to MGGA could reach up to 35.72%, which is a good result for this kind of transformation.