Abstract: Cordycepin was produced by semisolid fermentation of C. militaris using loofah as the attachment point of C. militaris spores and the adsorption matrix of vegetable oils in order to improve the specific surface area of liquid surface culture and further increase the cordycepin production. Single factor experiment and Box-Behnken experiment design were used to optimize the liquid content in loofah, spore inoculum size and fermentation temperature under the conditions of corn oil and olive oil by response surface methodology, respectively. The results showed that the optimum technological conditions of cordycepin were as follows: Corn oil as vegetable oil, liquid contents in loofah 3.0 mL/g, spore inoculum size 9.49%, fermentation temperature 27.3℃, relative humidity 90%. Under the optimized fermentation conditions, the cordycepin production reached 10.13 g/L, which was 76.48% higher than the initial semisolid fermentation. It suggested that, the control of fermentation temperature was the key factor to obtain high cordycepin production. Semisolid fermentation of C. militaris with loofah could obtain higher cordycepin accumulation by increasing specific surface area, which could provide theoretical support for large-scale industrial production of cordycepin.