Four new fluorescent dyes containing tetrahydro[5]helicene moiety characterized by three-primary emission colors (blue-green-red) are designed and synthesized, and their structures are characterized by NMR, MS, and single crystal X-ray crystallography. Organic nanoparticles based on the fluorescent dyes are then prepared by re-precipitation method, and their photophysical properties are investigated. These nanoparticles retain the strong emissions of the organic dyes, and multicolor nanoparticles were also prepared by simply tuning the ratios of the three-primary colors dyes. These organic nanoparticles exhibit low cytotoxicity, good photostability, and high quantum yields. Moreover, the nanoparticles can also be applied in the cell fluorescence imaging. Especially, it is interestingly found that the stained regions of these nanoparticles from membrane to cytoplasm for HeLa cells show obvious structure-dependent properties. This strategy provides a new perspective to fluorescence probe by molecular design for specific location imaging of living cells.