The invention relates to a protoplast mutation breeding method for improving the cordycepin content of cordyceps militaris. The method comprises the following steps of activated culture of bacterial strains, solid culture of mycelia, liquid mycelia culture, mycelia enzymolysis, protoplast nitrosoguanidine (NTG) mutation, protoplast regeneration culture, regenerated bacteria subculture, fermentation culture, filtering, further washing of mycelia, measurement of cordycepin content in the mycelia, preservation of the bacteria strain with high cordycepin content, measurement of the genetic stability and the like. Due to the adoption of the method, the cordycepin content of the cordyceps militaris can be increased.