<p>Cordycepin is a bioactive compound with demonstrated immunoregulatory, antioxidant, and organ-protective properties. This study aimed to significantly enhance the production of cordycepin in <i>Cordyceps militaris</i> fermentation through systematic process optimization. After identifying glucose, peptone, and Mg²⁺ as the optimal carbon source, nitrogen source, and inorganic salt via single-factor experiments, response surface methodology (RSM) was applied to refine their concentrations along with inoculation amount and pH. The analysis indicated that glucose, peptone, and pH exerted an extremely significant influence on yield (<i>p</i> &lt; 0.0001), Mg²⁺ was significant (<i>p</i> = 0.0112), while inoculation amount was not (<i>p</i> = 0.0524). The derived optimal conditions (40&#xa0;g L^− 1 glucose, 24&#xa0;g L^− 1 peptone, 0.7&#xa0;g L^− 1 Mg²⁺, 2% (v/v) inoculation, pH 8) were validated, achieving a cordycepin yield of 212.00 mg L^− 1. This result closely matched the model prediction and represented a 46.18% increase compared with the initial yield of 145.03 mg L^− 1, demonstrating the strategy’s effectiveness for boosting production efficiency and potential for industrial application. These results will help improve cordycepin production efficiency in industrial settings, thereby bringing economic benefits to the cordyceps industry.</p>