Mogrosides are the major triterpenoidal saponins found in swingle, the fruit of Siraitia grosvenorii, which have recently been widely used throughout the world as natural food sweeteners. Among this class of compounds, mogroside III E (MG III E) exhibits the most intense sweetness, and it was also found to effectively regulate blood glucose levels. However, the relative abundance of naturally occurring MG III E is low compared to other mogrosides. Therefore, the purpose of this study was to enrich MG III E through biotransformation of fruit extracts and to develop a reliable method for its purification. We used HPLC coupled with mass spectrometry and nuclear magnetic resonance spectroscopy for metabolite analysis and identified MG III E as a major metabolite of Ganoderma lucidum mycelium. This organism converts the most abundant mogroside, mogroside V, to MG III E via a deglycosylation reaction; high levels of β-glucosidase activities were also detected. In addition, we established an efficient purification method for MG III E using HP-20 macroporous resin. Optimization of the method was accomplished by kinetic model fitting, dynamic adsorption studies, and desorption experiments. The purity of MG III E was increased from 11.71% to 54.19%, with a 70%–76% recovery rate, and the scaled-up purification process allowed us to harvest 17.38 g of MG III E with 55.14% purity and a 74.71% of recovery rate. Therefore, our low cost, time-saving, easy to scale-up procedure for isolating MG III E could be applicable in industrial processes.