In the present work, a facile one-pot method is designed to fabricate a core-shell fluorescent nanoparticle (NP) for cellular imaging based on a new cationic conjugated polymer, poly[9,9'-bis(6,6'-(N,N,N-trimethylaminium)fluorene-2,7-ylenevinylene-co-alt-2,5-dicyano-1,4-phenylene] (PFVCN). Gold nanoflowers (AuNFs) are prepared by a seedless method, in which a gelatin layer formed through a sol-gel phase transition is deposited on the surface of each AuNF. The cationic PFVCN self-assembles onto the negative surface of the resultant (AuNF@Gelatin NPs) driven by electrostatic attraction. An obvious enhancement of fluorescence intensity is observed. The AuNF@Gelatin/PFVCN NPs exhibit excellent cytocompatibility, and their cellular imaging ability is demonstrated when cocultured with HeLa cells. AuNF@Gelatin/PFVCN hybrid NPs are expected to be a desirable material in the field of cellular imaging and biosensing.