For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35-5 g/L when its residual level decreased to 10-5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72+/-2.57 g DW/L, 7.09+/-0.62 and 4.43+/-0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.