Aero-optical effects in the mixing layer have caused significant concern due to the negative influence of high-speed vehicles with infrared imaging guidance systems. Here, we study the influence of different cooling mediums on the aero-optical effects. Four different cooling components are considered: helium (He), nitrogen (N2), air, and argon (Ar). The large eddy simulation method and ray-tracing method are used, respectively, to simulate the 3D mixing layer and to calculate the optical path difference (OPD). The numerical results show that, as the molecular weight of the cooling mediums increases, the mixing layer transition advances and the 3D effect of the flow field is enhanced. The local minimum OPD values of the wavefront distortion are significantly correlated with the large-scale vortex structure of the mixing layer. The compression effect plays a dominant role in aero-optics before the large-scale structure forms. Once the large-scale vortex structure generates, the aero-optical effect is conducted by the density and compression effect. The cooling medium helium delays the development of the mixing layer and subsequently reduces the aero-optical effects. However, once the large-scale vortex structure forms, the aero-optical effect becomes serious due to the largest density difference between air and He.