Polythiophene containing pendant N-hydroxysuccinimide (NHS) ester groups was reacted with poly(ethylene glycol) (PEG) and oleylamine, which was subsequently modified with folic acid to prepare a targeting fluorescent cellular imaging probe. The amphiphilic character of the folic acid-functionalized polythiophene induced the formation of nanoparticles with an average diameter of 95.3 nm in water. This polymer shows good photostability and low toxicity toward KB cells at a high concentration (100 µg/mL), which meets the crucial requirement for cellular imaging and other biological applications. The folic acid-functionalized polythiophene could be internalized efficiently into KB cells and accumulated in the cytoplasm to yield bright fluorescence, indicating that it is a good candidate material for the fluorescence imaging of living cells.