Abstract: Objective: The aggregation behavior of silk fibroin under specific conditions can influence its kinetic behavior and viscoelastic properties, thereby affecting biological properties of silk fibroin materials. Therefore, this study systematically investigated the self-assembly aggregation behavior of silk fibroin and explored the conditions for enhancing its mechanical performance and biological properties. Methods: Regenerated silk fibroin was extracted and prepared from domestic silkworm cocoons, and the optimal assembly conditions were determined through turbidity experiments. Rheological experiments were conducted to study the influence of Na⁺ on the kinetic behavior and viscoelastic properties of silk fibroin solutions. Cell proliferation experiments were carried out to investigate the biological properties of silk fibroin. Results: The extracted regenerated silk fibroin primarily exhibited a random coiled structure. The optimal in vitro self-assembly conditions involved assembly of a 4 mg/mL solution at 70 ℃ for 6 hours. The addition of Ca²⁺ to the silk fibroin solution inhibited in vitro self-assembly, while the addition of Na⁺ accelerated the assembly rate and increased the degree of assembly. Furthermore, with increasing Na⁺ concentration, the gel strength of silk fibroin gradually increased, leading to a more stable gel network. Cell proliferation experiments demonstrated that the solution of regenerated silk fibroin was more conducive to cell growth and proliferation than its self-assembled fibers. Conclusion: The concentration, temperature, assembly time, and ions all have an impact on the in vitro self-assembly aggregation behavior of silk fibroin. This study provides preliminary research on the self-assembly aggregation conditions and biological properties of silk fibroin, offering experimental basis for improving the performance of silk fibroin as a food packaging material.