To investigate the influence of the biaxial stretching process on the microstructural evolution and optical properties of polylactic acid (PLA) films, particularly the intrinsic correlation between the mesophase structure and the pearlescent effect, amorphous polylactic acid (PLA) cast sheets were prepared by melt extrusion casting, and biaxially stretched under different conditions to produce PLA films, with unstretched sheets used as controls. The effects of stretching temperature, ratio and rate on the content, morphology, average size, transmittance/haze, and pearlescence of spherical mesophases in PLA films were systematically investigated using Fourier transform infrared spectroscopy (FTIR), two-dimension wide-angle diffraction (2D-WAXD), polarized optical microscopy (POM), scanning electron microscopy(SEM), and light transmittance/haze meters. Results show that when stretched above the glass transition temperature but below 85?°C, increasing the stretch ratio and speed led to decreased transmittance, increased haze, and the development of a pearlescent effect. Optimal balance between opacity and pearlescence was achieved at 70–80?°C, 200–300?mm/s, and a stretch ratio of 3×3 to 3.8×3.8, where spherical mesophases were most uniformly distributed with an average size of 0.44–0.83?μm. Higher content and larger average size of the mesophases enhanced the pearlescent appearance of the films.