Silk derived from cocoons of the female silkworm (Bombyx mori) was made into three kinds of test material, degummed fiber, liquid fibroin and regenerated silk fibroin membrane (RSFM). The effect of the silk degumming and fiber dissolution system (SD–FDS) on the molecular structure and properties of silk fibroin were studied in detail. Degumming with Na₂CO₃ solution had the greatest impact on silk fibroin fiber, resulting in significant reduction of the thermal stability and crystallinity, and the fiber tensile property was only half of that degummed with urea buffer. The moderate degumming solution was urea buffer, followed by strongly alkaline electrolyzed water (SAEW). SDS-PAGE showed that the silk fibroin degummed with 8.0 M urea at 80 °C and dissolved in 9.3 M LiBr at 25 °C was similar to the natural silk fibroin in vivo, but boiling in Na₂CO₃ solution leads to serious breakage of the silk fibroin peptide chain. The degree of breakage of the peptide chain is positively correlated with the storage time of liquid silk fibroin. DSC showed the more nearly intact the silk fibroin peptide chain, the higher the thermal decomposition temperature. The regenerated condition of the silk protein is often overlooked during the research process, while our study showed that different silk degumming and fiber dissolution systems will have different influences. The results of this test help us gain a deeper understanding of the effect of silk degumming and fiber dissolution on the molecular structure and properties of silk fibroin and provide important experimental data for screening and using silk protein as advanced functional biomaterials, such as medical tissue engineering materials.