The aim of this paper is to obtain the single nucleus of Cordyceps militaris and the optimal preparation conditions of protoplasts. The number of cell nuclei of blastospores was observed by fluorescent staining. The optimized conditions for preparing protoplasts were studied by the single factor test and the Box-Behnken design with three variables as following： lywallzyme concentration， temperature， and digestion time. The yield of protoplasts was the evaluation index. The observation of fluorescence microscopy showed that the blastospores of Cordyceps militaris were single nuclei. The results of response surface optimization indicated that the optimal condition for preparing protoplasts was performed with 2.06% lywallzyme under 26.1 ℃ for 2.57 h. Under the optimal conditions， the predicted yield of protoplasts was 8.97×106 protoplasts per milliliter， whereas， the experimental yield of protoplasts was （9.17 ± 0.29）×106 protoplasts per milliliter. The predicted yield and the experimental yield of protoplasts were not significantly different from each other. The regeneration rate of protoplasts prepared under optimized conditions was up to 43.33% ± 2.08% on the regeneration medium containing 0.8 mol/L mannitol.