Brain-derived neurotrophic factor (BDNF) is a factor with many functions such as acceleration of cell proliferation and differentiation and is therefore widely used in clinical applications. In this study, an expression vector named pWYP23402 having a codon-optimized BDNF gene was constructed and transferred into chloroplasts of tobacco by gene-gun. After three or four rounds of selection with proper spectinomycin, BDNF was integrated into the chloroplast genome of homoplastomic plants confirmed by PCR and Southern hybridization. ELISA assay indicated that BDNF fused with GFP represented approximately 15.72%±0.33% of total soluble protein in the leaves of transplastomic plants. Moreover, the chloroplast-derived BDNF displayed similar biological activity to the commercial product. This is the first case report of BDNF expression by chloroplast transformation in this model plant, supplying an additional pathway for the production of chloroplast-expressed therapeutic proteins.