Monodisperse semiconductor colloidal spheres with a high refractive index hold great potential for building photonic crystals with a strong band gap, but the difficulty in separating the nucleation and growth processes makes it challenging to prepare highly uniform semiconductor colloidal spheres. Herein, real monodisperse Cu2O spheres were prepared via a hot-injection & heating-up two-step method using diethylene glycol as a milder reducing agent. The diameter of the as prepared Cu2O spheres can be tuned from 90 nm to 190 nm precisely. The SEM images reveal that the obtained Cu2O spheres have a narrow size distribution, which permits their self-assembly to form photonic crystals. The effects of precursor concentration and heating rates on the size and morphology of the Cu2O spheres were investigated in detail. The results indicate that the key points of the method include the burst nucleation to form seeds at a high temperature followed by rapid cooling to prevent agglomeration, and appropriate precursor concentration as well as a moderate growth rate during the further growth process. Importantly, photonic crystal films exhibiting a brilliant structural color were fabricated with the obtained monodisperse Cu2O spheres as building blocks, proving the possibility of making photonic crystals with a strong band gap. The developed method was also successfully applied to prepare monodisperse CdS spheres with diameters in the range from 110 nm to 210 nm.