Conjugated polymer nanoparticles hold promise as fluorescence imaging agents because of their high brightness and photostability. However, their aggregation and precipitation at the high ionic strengths in physiological conditions present an impediment to their applications in biological imaging. In this study, hydrophobic linear alkyl group substituted poly(acrylate)s, PAACn, are synthesized and employed as amphiphilic stabilizers. The carboxylate groups of PAACn increase the surface charge of conjugated polymer nanoparticles, stabilizing the particles in phosphate buffer saline, PBS. Nanoparticles of the green-yellow emitting conjugated polymer, F8BT, stabilized with PAACn (F8BT-PAACn) are prepared using nanoprecipitation. In contrast to the significant aggregation with a negligible yield (similar to 0%) of bare F8BT nanoparticles in PBS, high yields approaching 90% are observed for F8BT nanoparticles stabilized with PAAC18 at 1%, PAAC16 at 3%, and PAAC10 at 10% substitution. The F8BT-PAACn nanoparticles have a small size ranging from 50 to 70 rim in diameter, highly negative surface charge and high colloidal stability in PBS. These properties pave the way for the deployment of F8BT-PAACn nanoparticles in biological applications. Spectroscopic results indicate that PAACn has no adverse effect on the absorptivity and fluorescence brightness of F8BT-PAACn nanoparticles relative to bare F8BT nanoparticles. In addition, F8BT-PAACn nanoparticles are internalized by HEK 293 cells and exhibit low toxicity. In short, PAACn are versatile and robust stabilizing materials that facilitate the application of F8BT-PAACn nanoparticles as fluorescent probes in cell imaging.