Since nanodiamonds (NDs) exhibit low or no toxicity, NDs have attracted growing interest as promising materials for biomedical applications. Herein are reported size separation of ND particles by use of ultracentrifugation and synthesis of fluorescent ND through the surface chemical modifications. The convenient size separation method by use of ultracentrifugation gave desired sizes of NDs ranging from 4 to 25 nm in medium diameters by controlling the duration and acceleration of the centrifugation. The ND size is important for passive targeting such as EPR effect in their applications for DDS and tumor imaging. Surface chemical modification is also vital for adding requisite functions to NDs such as dispersibility and visibility. Step-wise transformations on ND surface were performed, providing fluorescent ND. The ND powder with 30 nm medium diameter was hydrogenated, followed by functionalization with aminoalkyl group via radical reaction. Fluorescent tag was immobilized onto the ND surface through amide linkage, which was confirmed by fluorescent image of the ND.