Antrodia cinnamomea (AC) has attracted much attention recently due to its pharmacological benefits, especially for anticancer, and immunomodulation effects. Triterpenoids are the major bioactive compounds present in AC, and responsible for cancer curing ability. Oxygen supply to fermentation is a crucial environmental parameter affecting triterpenoids production. Thus, 29.6 mg/g biomass dry weight (DW) of triterpenoids was obtained by static operation (limited oxygen supply) in the second stage of monoculture of AC. In this paper, we propose a novel approach to enhancing triterpenoids production of AC through two-stage fermentation by co-culture with Saccharomyces cerevisiae in the second stage under limited oxygen supply. As a result, 38.0 mg/g DW of triterpenoids was obtained in 250 ml shake flask. Triterpenoid content was further enhanced as 46.5 mg/g DW by increasing the inoculation level of S. cerevisiae to 25%. While a scale-up study of the novel fermentation strategy was executed in a 2 L airlift bioreactor, 62.2 mg/g DW of triterpenoids was achieved, which was 110% and 64% enhancement as compared to those of monoculture of AC in the flask and airlift bioreactor, respectively. This role of S. cerevisiae and its elicitors on triterpenoids production of AC were also investigated in this study.