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Cordyceps militaris is a medicinal mushroom in Asia in the 21st century, which cordycepin is a significant bioactive compound. This study, investigated the effect of culture conditions and vegetable seed extract powder as a supplementary source of animal-free nitrogen on the production of cordycepin by C. militaris in liquid surface culture. The highest cordycepin production was observed under soybean extract powder (SBEP) conditions, and 80 g L−1 of SBEP supplementation increased cordycepin production to 2.52 g L−1, which was greater than the control (peptone). Quantitative polymerase chain reaction was used to examine the transcription levels, and the results showed that supplementing with SBEP 80 g L−1 significantly increased the expression of genes associated with the carbon metabolic pathway, amino acid metabolism, and two key genes involved in the cordycepin biosynthesis (cns1 and NT5E) compared to peptone-supplemented culture. Under optimal culture conditions, the model predicted a maximum response of cordycepin production of 2.64 g L−1 at a working volume of 147.5 ml, an inoculum size of 8.8% v/v, and a cultivation time of 40.0 days. This optimized culture condition could be used to increase cordycepin production in large-scale bioreactors. Additional research can be conducted to assess the economic viability of this process. [ABSTRACT FROM AUTHOR]