Tobacco mosaic virus (TMV) is an important virus pathogen that threatens the production of tobacco. In the past 50 years, only the N gene has been deployed for TMV-resistant tobacco breeding, while the TMV-resistant flue-cured tobacco varieties carrying the N gene are difficult to be promoted due to the linkage drag. At the same time, the single-gene dominant resistance is at risk of being overcome by the virus. Therefore, the development and utilisation of new resistance genes is of great significance. The N'alata gene from wild tobacco Nicotiana alata, which is the N' orthologs from cultivated tobacco, is a TMV-resistance gene and shares a high sequence identity with the N' gene, which has no resistance to TMV. By performing a series of fragments swap between N'alata and N' genes, we confirmed that the 265-798^th amino acids and the 1130-1233^th amino acids of N'alata jointly determined its resistance to TMV-U1. Therefore, gene editing can be used to directly modify the corresponding functional region of N' in cultivated tobacco, so that the main cultivar can acquire TMV resistance and maintain the original agronomic and economic traits. This study thus combined the gene insertion method reported in rice and tobacco protoplast technology, and preliminarily achieved gene insertion and replacement in tobacco protoplasts. Afterwards, aiming at the biggest difficulty in the protoplast system—protoplast regeneration of plants, we carried out a series of optimisations for protoplast isolation, transfection and regeneration, and obtained a stable and efficient procedure of protoplast regeneration. Finally, the system of protoplast-mediated insertion and replacement of tobacco genes was established. In this study, the established gene knock-in system was used to replace the corresponding segment of the N' gene in cultivated tobacco with N'alata resistance region. The replacement of the cultivated tobacco gene was achieved and conferred resistance to TMV. The tobacco gene insertion and replacement system established in this study can be widely used in tobacco breeding research, which could promote the development of tobacco genome editing research.