During crystallization, conformational changes are often accompanied by the formation of interactions. Terahertz (THz) spectroscopy exhibits strong responses to the crystalline poly(lactic acid) (PLA). Therefore, we estimate the relative crystallinity and investigate the effect of conformational transition on the vibration of PLA by THz spectroscopy. By comparing with the results of X-ray diffraction (XRD) and differential scanning calorimetry (DSC), the validity of THz spectroscopy to calculate crystallinity is verified. Furthermore, the peak intensity of PLA at 2.01 THz increases with crystallinity. Combined with Fourier transform infrared spectroscopy (FTIR), the vibrational intensity of PLA at 2.01 THz is highly correlated with the contribution of gt conformation, showing a linear relationship. In addition, the vibrational peak of PLA also reflects the interchain interactions. We believe that the increase in peak intensity with increasing crystallinity originates from the effect of the dipole−dipole interactions between the carbonyl groups. Our study demonstrates the ability of THz spectroscopy to estimate the crystallinity of PLA, and the peak at 2.01 THz shows conformational and interaction sensitivities.