The objectives of this research were to determine the optimum fermentation condition and to enhance the 1,3-β-D-glucan production of Ganoderma lucidum in submerged culture. The immunopotentiation of the polysaccharide from G. lucidum fermentation was evaluated by the inhibitory ratio of leukemic U937 cell as an model. The basic medium consisted of 5% black soybean, 2% Huangqi and 2% glucose. Furthermore, 1% glucose was added on day 4, 6 and 8, respectively. The effect of feeding time during the 12-day cultivation in 5L fermentor was evaluated. Addition of 1% glucose on day 6 inhibited the 1,3-β-D-glucanase activity and enhanced 1.54-flod 1,3-β-D-glucan in the culture. Moreover, the addition of chitin synthase inhibitor, Polyoxin B (1.5 ppm), probably reduced the binding between the structure glucan and chitin in cell wall, and therefore enhanced 1,3-β-D-glucan content up to 1.68 fold on day 8 without affecting cell growth. The inhibitory ratio of leukemic U937 cell resulted from the G. lucidum polysaccharides was positive correlated to the percentage of 1,3-β-D-glucan, molecular weight and degree of branching. Moreover, significant increases of TNF-α, IL-1β and IFN-γ in the mononuclear condition medium treated with the polysaccharidrs of 1% glucose fed-batch on day 6 were observed.