Extracellular lipase from Antrodia cinnamomea BCRC 35396 was first purified by ammonium sulphate precipitation and DEAE-Sepharose chromatography. The yield and purification factor were 33.7% and 17.2 folds, respectively. Lipase production from A. cinnamomea was enhanced by 0.01% olive oil supplementation as additional carbon source in an aerated bioreactor with final titer 26 U/ml after 18 days of fermentation. The molecular weight of the purified enzyme was estimated to be 60 kDa by SDS-PAGE. The enzyme was found to be alkaline tolerant (pH 7-10) with optimum activity at pH 8.0, but both activity and stability decreased significantly as pH value greater than 10. The enzyme activity was clarified to be stable within the temperature range of 25-60°C, with maximal activity at 45°C. This observation was similar to those of mesophiles as expected.