A general strategy for the preparation of highly fluorescent poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (NPs) loaded with conjugated polymers (CPs) is reported. The process involves encapsulation of organic-soluble CPs with PLGA using a modified solvent extraction/evaporation technnique. The obtained NPs are stable in aqueous media with biocompatible and functionalizable surfaces. In addition, fluorescent properties of the CP-loaded PLGA NPs (CPL NPs) could be fine-tuned by loading different types of CPs into the PLGA matrix. Four types of CPL NPs are prepared with a volume-average hydrodynamic diameter ranging from 243 to 272 nm. The application of CPL NPs for bio-imaging is demonstrated through incubation with MCF-7 breast cancer cells. Confocal laser scanning microscopy studies reveal that the CPL NPs are internalized in cytoplasm around the nuclei with intense fluorescence. After conjugation with folic acid, cellular uptake of the surface-functionalized CPL NPs is greatly enhanced via receptor-mediated endocytosis by MCF-7 breast cancer cells, as compared to that for NIH/3T3 fibroblast cells, which indicates a selective targeting effect of the folate-functionalized CPL NPs in cellular imaging. The merits of CPL NPs, such as low cytotoxicity, high fluorescence, good photostability, and feasible surface functionalization, will inspire extensive study of CPL NPs; as a new generation of probes for specific biological imaging and detection.