A two-stage submerged fermentation with elicitation was used to increase triterpenoids production of Antrodia cinnamomea in an airlift bioreactor with dual net draft tube. The dual net draft tube bioreactor had higher kLa value (7.3h−1) than the solid tube bioreactor (5.5h−1). The fungus was cultured at 25°C for 12days, then elicited with chemicals and physical factors. It was found that comparing to calcium chloride, chitosan was a more potent chemical elicitor. At dosage level of 100mg/L, it not only resulted in more biomass production but also yielded higher crude triterpenoids production. The triterpenoids production could be significantly enhanced by means of the oxygen limitation and temperature fluctuation strategies. The maximum triterpenoids production could be achieved by using static culture (oxygen limitation) with temperature fluctuation between 25°C (for 10h) and 10°C (for 14h). The in vitro anticancer activity assay indicated that the crude triterpenoids produced by A. cinnamomea using the developed elicited two-stage cultivation protocol effectively inhibited proliferation of human cervical epithelioid carcinoma HeLa cells in a dosage dependent manner. The IC50 of the crude triterpenoids from the mycelium of A. cinnamomea after 21days cultivation for HeLa cells was 25μg/mL.