Conjugated polymers possess excellent qualities as fluorescent probes for biomedical applications, because of their extremely high brightness, extinction coefficients, and photostability. Encapsulating these hydrophobic polymers in nanoparticulate form allows transfer to aqueous environments and construction of high-performance fluorescent nanoparticle constructs, and several surface capping strategies have been demonstrated to date. Here, we describe the development of a new class of multifunctional capping ligands for conjugated polymer nanoparticles based on custom-designed amphiphilic peptides. These versatile peptide ligands provide a protective hydrophilic capping layer, chemical handles for further conjugation, and directed biological activity tuned by altering the specific amino acid sequence. We show that (i) cellular uptake can be regulated as a function of peptide composition, and (ii) the nanoparticles show no signs of toxicity under the conditions used, which is a vital health and environmental issue when developing these technologies for clinical use. Finally, we demonstrate that this one-pot method can be applied can be applied to three classes of conjugated polymers and demonstrate potential for multicolor imaging.