Abstract: Binary colloidal crystals were fabricated by a vertical deposition method with two sub-micrometer size polystyrene (PS) microspheres. The self-assembled patterns of binary colloidal crystals (bCCs) were investigated by varying the size ratio, the mass ratio of PS microspheres, the temperature and content of ethylene glycol in water. It is found that PS binary colloidal crystals with face-centered cube (fcc) arrangement can be obtained while the size ratio is more than 4.45 and the mass ratio is not less that 74:26. High-quality PS binary colloidal crystals are easily generated at the temperature of 60℃ and the mass fraction of ethylene glycol in dispersing medium of 6%. The study also shows that self-assembly of two PS microspheres on the substrate surface is operated at the same time, and in this regard the vertical deposition method has a great deal of difference from other methods. In addition, PS binary colloidal crystals have a higher quality with the increase of matching between Brownian motion, sedimentation rate of PS colloidal microspheres in water, and self-assembly rate on the substrate surface.