The fibroblast is one of the ideal target cell candidates for cell-based gene therapy approaches to promote tissue repair. Gene delivery to fibroblasts by viral transfection has been confirmed to have high transfection efficiency. However, in addition to immunogenic effects of viruses, the random integration of viral genes may damage the genome, affect the cell phenotype or even cause cancerous mutations in the transfected cells. Due to these potential biohazards and unknown long-term risks, the clinical use of viral transfection has been very limited. In contrast, initial non-viral transfection methods have been simple and safe to implement, with low immunogenicity, insertional mutagenesis, and risk of carcinogenesis, but their transfection efficiency has been relatively low. Nucleofection, a more recent non-viral transfection method, now combines the advantages of high transfection efficiency and direct nucleic acid delivery to the nucleus with a high safety. Here, we reviewed recent articles on fibroblast nucleofection, summarized different research points, improved methods and application scopes, and opened up ideas for promoting the further improvement and development of fibroblast nucleofection to meet the needs of a variety of disease research and clinical applications.