Abstract: To further improve the cordycepin production, the effects of three plant growth regulators, triacontanol, gibberellin and 6-benzylaminopurine, and their combinations on mycelial growth of Cordyceps militaris and cordycepin accumulation were investigated in the surface liquid fermentation. Through central composite design and response surface method, a mathematical model was developed to identify the optimal value of plant growth regulators combination for high cordycepin production. The results showed that the three plant growth regulators can promote the growth of Cordyceps militaris and cordycepin accumulation to different degrees. When the three were used respectively, gibberellin exhibited the best growth promoting effect and the optimum concentration was 10 mg/L. The triacontanol exhibited the best cordycepin-producing effect and the optimum addition was 0.6 mg/L. The optimal combination of 0.55 mg/L triacontanol, 22.64 mg/L gibberellin and 1.69 mg/L 6-benzylaminopurine was the most favorable to cordycepin accumulation. Under the optimal conditions, the maximum cordycepin yield reached 7.31 g/L.Therefore, the combination of the plant growth regulators was favourable for cordycepin accumulation