1-Deoxynojirimycin (DNJ), an alpha-glucosidase inhibitor, has been used to prevent or treat type 2 diabetes. Low amounts of DNJ are found in mulberry leaf; however, a methodology is necessary to enhance the DNJ content of mulberry leaf extract (MLE) since increasing the amounts of DNJ is required for the prevention and treatment of type 2 diabetes. In this study, the DNJ content of MLE was increased through the optimization of the conditions for MLE fermentation by Lactobacillus plantarum SG-053, using response surface methodology (RSM). By single factor testing, the optimal conditions were identified as an inoculum concentration of 1%(v/v), an MLE concentration of 3°Bx, and no agitation. Based on a Plackett-Burman design, the following factors were considered to majorly affect the DNJ content in the MLE fermentation product: the initial pH, fermentation temperature, and fermentation time. By response surface methodology, the optimal conditions for MLE fermentation was found to have an initial pH of 5.5, a fermentation temperature of 31.9℃, and a fermentation time of 34 h. Under these optimal fermentation conditions, the DNJ content in MLE increased 3.59 times, or from 23.85 to 85.54 μg/mL.