Uniform and monodispersed ZnSe colloidal microspheres were prepared via a hot injection route by using trioctylamine as solvent and with a relatively higher concentration of the monomer precursors. The size of the colloidal ZnSe spheres was tuned from 138 to 629 nm by varying the concentration of the precursors, and the size increased linearly with the concentration of Zinc oleate in the range from 0.05 to 0.10 kg/L with the molar ratio of Zn to Se of 0.5. The colloidal microspheres were well characterized by SEM, TEM, HRTEM, EDS, UV-vis, and PL techniques. Detailed examination confirmed that the colloidal microspheres were formed by the in-situ aggregation of ZnSe nanoparticles. Furthermore, the 2D ordered assembly of the ZnSe microspheres was realized via a simple vertical precipitation technique. Due to the high refractive index and absence of absorption in the visible region, the monodispersed ZnSe colloidal microspheres could be potential building blocks to construct photonic band gap crystals and other functional devices.