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Nerve growth factor (NGF) is one of the most critical neurotrophic factors in the nervous system and plays an important role in regulating neuronal growth, development, differentiation, and survival, as well as the regeneration and repair of damaged nerves. Currently, the main active component of pharmaceutical NGF products in China is mouse-derived nerve growth factor (mNGF). However, mNGF may cause allergic reactions and carries the risk of animal virus contamination. This study used the tobacco BY-2 cell expression platform to produce bioactive recombinant human nerve growth factor (rhNGF), aiming to overcome the limitations associated with mNGF. The human NGF gene was introduced into BY-2 cells <i>via</i> genetic engineering, and PCR analysis confirmed its integration into the cell genome. Western blotting results demonstrated that rhNGF was successfully expressed at 166 μg/L in BY-2 cells, and the recombinant protein induced PC12 cell differentiation and promote TF1 cell proliferation, confirming its favorable biological activity. The results of this study paves the way for the use of tobacco BY2 cells as an effective production system for active human nerve growth factor. Our work not only provides a feasible plant-based expression strategy for obtaining safe and low-immunogenicity human NGF, but also lays a technical foundation for utilizing plant cell platforms to produce other complex therapeutic proteins.