Abstract: The paper aimed to analyze the principal factor of affecting dissolved oxygen in the shaking culture of Cordyceps sinensis mycelia.Signal-factor test was carried out by measuring the yield of Cordyceps sinensis mycelia on different shaker speeds,liquid volumes and sealing modes,and then three factors of orthogonal test was carried out.Results showed that the appropriate conditions of dissolve oxygen were as follows：the shaker speed was 140 r/min;the liquid capacity was 50% and the seal mode was to use sealing film of aseptic culture vessel.The influencing significance of factors was in order of shaker speed,liquid capacity and seal mode.Determining result of active ingredient content under different shaker speeds showed that the shaker speed had no significant influence on the polysaccharide and oxalic acid contents of Cordyceps sinensis.