<p>Cordycepin is an important quality control component in <i>Cordyceps militaris</i>. As a result of its known biological activities, cordycepin from <i>Cordyceps militaris</i> has been extensively characterized in regards to potential medicinal applications. Therefore, optimized liquid culture conditions for enhanced cordycepin productivity have been developed, which are necessary steps for industrial applications. In this study, the effects of cottonseed powder and perilla oil as secondary nitrogen and carbon sources on promoting cordycepin production from <i>Cordyceps militaris</i> were evaluated. The proper amount of cottonseed power and perilla oil was optimized using response surface methodology. The results showed that the optimal medium (initial pH 5.7) contained 20.61&#xa0;g L^− 1 cottonseed powder and 8.28&#xa0;g L^− 1 perilla oil. Under these conditions, the cordycepin production yield of <i>Cordyceps militaris</i> reached 1.49 ± 0.04&#xa0;g L^− 1, which was about 1.602-fold that of the control group without cottonseed powder and perilla oil (0.93 ± 0.03&#xa0;g L^− 1). The results showed that cottonseed powder instead of expensive yeast extract could be used as nitrogen source and perilla oil could effectively improve the yield of cordycepin from <i>C. militaris</i>.</p>