Spider silk plays a pivotal role in the diverse physiological activities of spiders, with its protein components exhibiting remarkable mechanical properties and biocompatibility. Spider silk proteins exhibit a high degree of repetitiveness, primarily constructed through the recurring arrangement of amino acid motifs, including (A), (GA), (GGX), and (GPGXX) sequences. These repetitive sequences endow spider silk with different material properties. Recombinant spider silk proteins are produced through heterologous expression systems, and then spun into nanofibers using artificial spinning technology. These fibers have broad potential applications in the biomedical field, such as tissue engineering scaffolds, drug delivery carriers, sutures, and other biomaterials. However, enhancing the yield and performance of recombinant spider silk proteins, while facilitating large-scale production, continues to pose a significant challenge in the current landscape.