A novel method using Plackett-Burman design (PBD) coupled with Draper-Lin small composite design (SCD) and desirability function (DF) was developed to optimize Tuber melanosporum fermentation medium. Firstly, sucrose, yeast extract, peptone and Mg(2+) were identified to be key medium components by PBD. Secondly, in order to evaluate the relationships between responses and the identified key components, mathematical models were developed by SCD. Finally, by using DF, the responses were optimized simultaneously and the optimal concentration was located to be 73 g/L sucrose, 11 g/L yeast extract, 8 g/L peptone, and 46 mM Mg(2+). Under the identified optimal conditions, the maximal biomass, the production of extracellular (EPS) and intracellular (IPS) polysaccharides was 25.10 +/- 0.12 g/L, 3.88 +/- 0.23 g/L and 2.87 +/- 0.32 g/L, respectively, which agreed with the predicted Values well. Compared with the basic medium, the biomass, the production of EPS and IPS was enhanced by 54.4%, 71.7% and 124.2%, respectively. (C) 2009 Elsevier Ltd. All rights reserved.