During the submerged fermentation of medicinal mushroom Chinese truffle Tuber sinense, there was no significant effect of metal ion on the cell growth and the production of intracellular polysaccharides, while metal ion and its concentration significantly affected the production of extracellular polysaccharides (EPS). By using the approach of “one-variable-at-a-time”, 50mM Mg 2+ was identified to be the most favorable for EPS production, and the next was 10mM K + . A mathematical model, constructed by response surface methodology combination with full factorial design, was applied to study the synergic effect of Mg 2+ and K + . EPS production reached its peak value of 5.86g/L under their optimal combination of 30mM Mg 2+ and 5mM K + predicted by the model, which was higher by 130.7% compared with the basal fermentation medium without metal ion. The validation experiment showed the experimental values agreed with the predicted values well. EPS production obtained in this work was the highest reported in the culture of T. sinense.