Abstract: Polarized light microscopy and Raman spectroscopy were used to study the effects of shear rate and Ca²⁺ ions on the conformational transition of the aqueous solution of regenerated silk fibroin. The results showed that the shear rate and Ca²⁺ ions are really the two main influencing factors. For the same regenerated solution, the higher the shear rate, the more the silk fibroin molecules in orderly arrangement along shear rate in the solution. Then, the solution is easier to be transferred to β-pleating conformation from irregular coil or α-helix one, and such a transition is strengthened with increasing shear rate. After dialysis the Ca²⁺ ion concentration in the solution increases, and the critical shear rate with which the polarized light is visible decreases to a certain extent. And the Raman spectra revealed that the molecular conformation of the silk fibroin is transferred to Silk II from Silk I.