Three fluorescent amphiphilic rod-coil grafted copolymers comprising fluorene-based backbones and PEO side chains have been synthesized through the Suzuki coupling reaction. Stable and uniform fluorescent micelles were formed from the fluorescent amphiphiles in aqueous solution. The micelle size was tuned from 85 to 178 nm through controlling the hydrophilic/hydrophobic ratio and molecular weight of the amphiphiles. Preliminary biocompatibility and bioimaging investigation with BV-2 cells indicates that the fluorescent micelles are noncytotoxic, and BV-2 cells can be uniformly labeled by the micelles. Because of the intense fluorescence, biocompatibility, and noncytotoxicity of the amphiphiles, the fluorescent micelles may find potential applications in visualization of microscopic structures and drug delivery tracing.