Silk fibroin extracted from the gland of Bombyx mori silkworm is employed as an ideal system to investigate its conformation transition in methanol/D₂O solution. The transition process was monitored by Fourier transform infrared (FTIR) spectroscopy coupled with terahertz time domain spectroscopy (THz-TDS). Analysis of FTIR spectra suggests that, with increasing time of treatment, an increasing band at 1634 cm⁻¹ is observed indicating the formation of β-pleated sheets coincident with the loss of intensity of a band at 1673 cm⁻¹ indicating decrease of the random coil structure. In addition, there is a burst phase of 33% occurring during the first 2 minutes when the gland fibroin membranes are immersed into methanol/D₂O solution. THz spectra present distinct features for conformations of silk fibroin, in combination with the results obtained from FTIR; the peaks observed at 1.54 THz (51 cm⁻¹), 1.67 THz (55 cm⁻¹), and 1.84 THz (61 cm⁻¹) can be attributed to a β-pleated sheet, α-helix, and random coil, respectively. Intensity change of bands centered at 1.54 THz and 1.84 THz confirms the formation of the β-pleated sheet and the disappearance of the random coil. Kinetic curves obtained from THz spectra indicate that the methanol-induced conformation transition from the random coil to the β-pleated sheet is fitted with an exponential function. The results suggest that THz-TDS presents great potential as a complementary approach in studying the secondary structure of a protein, providing significant insight into the silk-spinning process in vivo.