Ordered silica nanosphere templates, which are usually known as colloidal crystals, are most widely used to prepare ordered porous nanostructure materials among the templates to fabricate nanostructure materials. We present here a method for the simultaneous assembly of multiple ordered silica nanosphere templates with same quality, in which a glass trough together with a stand was used as the experimental cell. Two different diameters of silica colloidal particles were selected for our experiments, namely ∼190 nm and ∼280 nm. The growth process, thickness and optical properties of films of silica nanospheres on substrates were studied. The particle sedimentation and solvent evaporation both play a role in determining particle volume fractions. In addition, the standard deviation of the diameter of the particles affects the optical properties of the films along the growth direction. There was almost no difference observed in our measurements of the film thickness and optical properties for both the same regions of different films and different regions of the same film along the direction perpendicular to the growth direction. The elucidation of the growth process and characterization of the film properties achieved in this study could help us to obtain better quality templates. This is the first systematic study of the evolution of the thickness and optical properties of ordered silica nanosphere films formed by a simultaneous assembly process.