Biochar, a soil conditioner, has the potential to improve soil properties and plant productivity. However, in forestry planting, especially in subtropical moso bamboo forests, the response of seedling growth to biochar addition is still not well known. We conducted a comprehensive factorial experiment with biochar and nitrogen (N) addition as factors (no biochar and no N addition; 0.64% biochar + 0% NH4NO3; 1.28% biochar + 0% NH4NO3; T3: 0% biochar + 1.28% NH4NO3; T4: 0.64% biochar + 1.28% NH4NO3; T5: 1.28% biochar + 1.28% NH4NO3) to study their effects on moso bamboo seedling growth, rhizosphere soil nutrient contents, and enzymatic activity. Our results indicate that applying biochar without N did not promote the growth of moso bamboo seedlings (biomass of leaves and branches) but increased soil nutrient content and affected soil-enzyme activity. The combined application of biochar and N significantly increased the leaf and branch biomass of moso bamboo seedlings and soil nutrient content and affected soil-enzyme activity. In conclusion, biochar should be mixed with an adequate amount of N for its application in subtropical moso bamboo forests to promote seedling growth and improve economic benefits.