Abstract: The Plackett-Burman experiment, the steepest ascent experiment and the central composite design were used to optimize the fermentation medium for the production of geraniol by the recombinant E.coli. Firstly, Plackett-Burman design was used to screen the critical factors which influenced the geraniol production from seven mediums. Secondly, the method of steepest ascent was adopted to make the key factors approach the optimal response area. Finally, the optimal concentration of key factors and regression model were obtained by central composite design. It was indicated that ammonium ferric citrate, MgSO4, and trace element solution were identified to be the significant factors and the it optimal concentrations were 0.13 g/L, 2.12 g/L, and 16.7 mL/L, respectively. The geraniol concentration increased to 223.24 mg/L under these conditions. It was about 3.26 times more than that of before.