The effects of aeration rate, agitation rate and the impeller type on the L-DOPA production by S. hassjoo cells were investigated in a 5L agitated bioreactor. Reactor performance was assessed by measuring K(L)a, DO, mixing intensity (shear stress), off-gas composition, specific oxygen uptake rate (SOUR) and specific CO2 evolution rate (SCER), The aeration rate played an important role in cell growth and secondary metabolite production. The K(L)a, mixing characteristics and shear stress depended on impeller design. A dual impeller system consisting of a flat-blade turbine and pitched-blade turbine provided a high oxygen transfer rate that was beneficial to the cell growth and L-DOPA production. At 0.06 vvm and 300 rpm, the dual impeller system supported high cell growth rate and L-DOPA content with an average growth rate and growth index similar to shake flask production. The operation strategy for S. hassjoo suspension culture is to determine the aeration rate that allows the sufficient DO and pertinent gas composition to be maintained. Enhancement of oxygen transfer rate at an acceptable shear stress by incorporating a specific impeller and adjusting the agitation rate was essential for successful cell culture in the agitated bioreactor.