This work reports a newly developed two-step hydrothermal method for the synthesis of monodispersed colloidal carbon spheres (CCS) under mild conditions. Using this approach, monodispersed CCS with diameters ranging from 160 to 400 nm were synthesized with a standard deviation around 8 . The monomer concentration ranging from 0.1 to 0.4 M is in favor of generation of narrower size distribution of CCS. The particle characteristics (e.g., shape, size, and distribution) and chemical stability were then characterized by using various techniques, including scanning electron microscopy (SEM), FT-IR spectrum analysis, and thermalgravity analysis (TGA). The possible nucleation and growth mechanism of colloidal carbon spheres were also discussed. The findings would be useful for the synthesis of more monodispersed nanoparticles and for the functional assembly.