Conjugated polymers are versatile bio-imaging probes as their optical properties can be readily fine-tuned. In this manuscript, fluorescent conjugated polymer nanoparticles are fabricated using three different poly(p-phenylene ethynylene) (PPE) derivatives. The polymers have the same backbone but carry different side chains, i.e. regular octyloxy substituents, half of the octyloxy chains azide terminated, or azide functionalized tetraethylene glycol (TEG) moieties. The azide groups are specifically chosen to allow coupling of (bio)molecules to the surface of the particles using straightforward azide-alkyne click reactions, enabling different bioconjugation and targeting strategies. The influence of the functionalization pattern on the size and optical properties of the nanoparticles is studied using transmission electron microscopy, dynamic light scattering, UV-Vis absorption and fluorescence spectroscopy. The polymer containing the azide functionalized TEG chains affords larger particles, which can be attributed to hydration of the outer layer of the more hydrophilic polymer particles. However, this does not impact the fluorescence quantum yield. The two azide functionalized PPE particles exhibit the highest quantum yields (13%). Despite the presence of azide groups on two of the three materials, all particles are biocompatibre and taken up by A549 human lung carcinoma cells. A proof of concept click reaction was performed as well. (C) 2017 Elsevier Inc. All rights reserved.