Cordyceps militaris stromata on Antheraea pernyi pupae contain various active components, including cordycepin, adenosine, polysaccharides, and amino acids. Response surface methodology (RSM) was used to optimize the liquid culture conditions for C. militaris before its injection into A. pernyi pupae. A pH of 7.56 ± 0.02, a culture temperature of 20.5 ± 0.1 °C, a culture time of 110.5 ± 0.5 h, and a KH 2 PO 4 concentration of 1.11 ± 0.01 g l −1 resulted in a C. militaris dry weight of 1.0226 g l −1 . Experimental and predicted values were similar. The RSM optimization increased the number of fruiting bodies (17 to 22) and the average fruiting body length (6.9 cm to 7.9 cm), while also deepening the yellow colouration of the fruiting bodies. The adenosine, cordycepin, polysaccharide, carotenoid, and cordycepic acid contents increased by 12.52%, 7.67%, 3.03%, 14.93%, and 0.02%, respectively, after the optimization. However, the optimization did not alter the number of different amino acids (18) or the total amino acid content, even though the contents of certain amino acids changed somewhat. These finding may be useful for increasing the yield of C. militaris stromata on A. pernyi pupae, which will increase the profitability of C. militaris production.