There is currently a high demand for novel approaches to engineer fluorescent nanoparticles with precise surface properties suitable for various applications, including imaging and sensing. To this end, we report a facile and highly reproducible one-step method for generating functionalized fluorescent organic nanoparticles via self-assembly of prefunctionalized pi-conjugated oligomers. The engineered design of the nonionic amphiphilic oligomers enables the introduction of different ligands at the extremities of inert ethylene glycol side chains without interfering with the self-assembly process. The intrinsic fluorescence of the nanoparticles permits the measurement of their surface properties and binding to dye-labeled target molecules via Forster resonance energy transfer (FRET). Co-assembly of differently functionalized oligomers is also demonstrated, which enables the tuning of ligand composition and density. Furthermore, nanoparticle prefunctionalization has been combined with subsequent postmodification of azide-bearing oligomers via click chemistry. This allows for expanding ligand diversity at two independent stages in the nanoparticle fabrication process. The practicability of the different methods entails greater control over surface functionality. Through labeling with different ligands, selective binding of proteins, bacteria, and functionalized beads to the nanoparticles has been achieved. This, in combination with the absence of unspecific adsorption, clearly demonstrates the broad potential of these nanoparticles for selective targeting and sequestration. Therefore, controlled bifunctionalization of fluorescent pi-conjugated oligomer nanoparticles represents a novel approach with high applicability to multitargeted imaging and sensing in biology and medicine.