A new fluorescent nanoparticle based on lipid-modified cationic poly(fluorenylene phenylene) (PFPL) was prepared and characterized. The PFPL is amphiphilic and has two unique structural features. First, the lipid is designed as a side chain in consideration of the fact that the lipid is biocompatible and can enter the cytoplasm easily and also provides protective layers to conjugated polymer backbones. Second, the ammonium pendant groups can interface with genes that offer PFPL as a vector for gene delivery. The PFPL forms uniform nanoparticles in water with a size of about 50 nm. The PFPL nanoparticle exhibits excellent photostability and little cytotoxicity. It can easily enter the cytoplasm via endocytosis (within 4 hours) and can be used for cell imaging. Furthermore, the PFPL particle can successfully deliver plasmids (pCX-EGFP) into lung cancer cells (A549) for transcription and translation. Thus, one can directly track the entrance of plasmids into cells and the expression of GFP proteins using fluorescence microscopy. The PFPL nanoparticle is a good candidate for cell imaging and tracking important intracellular processes in future research.