Abstract: In order to seek the technological process of large-scale cordycepin production, the fermentation medium and conditions for Cordyceps militaris CICC No.14014 to produce cordycepin in static fermentation were optimized. Based on the study of single factor tests, the optimal inoculation method was achieved with 3d seed age and 10% (v/v) inoculum size to improve the mathematic statistics precision and cut down the fermentation period. We used Plaekett-Burman design to evaluate the influence of seven related factors.Fermentation temperature and concentrations of yeast extract and tryptone were affirmed the significant factors.After identifying three critical factors, the optimal region of cordycepin production was found by steepest ascent approach. Then central composite design and response surface methodology were also used to develop a mathematical model to identify the optimum values of the key factors for higher cordycepin production, and to confirm its experimental validity. Results showed that the optimal fermentation medium and conditions were as follows:glucose 60g/L, KH2PO40.7g/L, MgSO4·7H2O 0.7g/L, yeast extract 9.00g/L, tryptone 17.10g/L, the initial pH6.30, fermentation temperature 27.1℃. Under these optimum conditions, the yield of cordycepin reached6.50g/L, which led to a 2-fold increase compared with that before.