Hybridbio/‐synthetic sensory conjugated polymer nanoparticles (CPNs) are developed for selective label‐free detection of target ssDNA in serum. Carboxylic acid‐functionalized anionic polyfluorene nanoparticles are rationally designed as signal amplifying unit to bioconjugate with amine functionalized single stranded oligonucleotides as a receptor. The covalent DNA coating can significantly improve the photostability of the DNA‐bioconjugated CPNs over a wide range of buffer conditions. Better ssDNA discrimination for the DNA‐bioconjugated CPNs sensor is achieved owing to increased interchain interactions and more efficient exciton transport in nanoparticles. The distinguishable fluorescent color for DNA‐bioconjugated CPNs in the presence of target ssDNA allows naked‐eye detection of ssDNA under UV irradiation.