Chitosan is a plant growth regulator. Since it has an important function, it may also play a similar role in the growth of fungi. In this study, Lentinula edodes was used as a model fungus, and the mycelium growth and metabolism in the cultivation bag treated with different concentrations of chitosan were investigated. The results showed when the chitosan concentration was 0.1 mg/ml, a dense mycelium was developed and the fast growth was observed. Compared with other concentrations and the control group, they had significant differences. Furthermore, the activity of CMCase, Amy, and POD were measured, which showed significant differences in the rapid growth of the mycelium compared with other concentrations. The concentrations of 0.1 mg/mL will be chosen to promote the growth of Lentinula edodes' mycelium in practical production.