As emiconductor polymer with side-chain oxetane groups was synthesized to form highly fluorescent, functionalized, andu ltra-stablep olymer dots (Pdots). A facile light-induced strategy covalently links functional polyethylene glycol (PEG) molecules to polymer dots while simultaneously providing functional groups for bioconjugation. The influence of photo-crosslinkable sidechains and PEG molecules on the properties and performance of Pdots was systematically investigated. The Pdots functionalized by photo-crosslinking show specific labeling and negligible non-specific binding as compared to non-functionalized Pdots. The resultsp rovide av iable strategy for nanoparticle assembly and functionalization.Semiconducting polymers have attracted considerable interest in both optoelectronic and biomedical applications over past decades. While they were originally developed for optoelectronic devices,t he adaptation of semiconducting polymers into nanoparticles has yielded ar apidlyi ncreasing number of applicationso ft he polymer dots (Pdots)i nb iomedical theranostics.[1-4] The attractiveness of Pdots foro pticali maging, sensing, and phototherapy is due to their superior characteristics, such as large absorption coefficients, excellent photostability,b iocompatibility,a nd suitability for the efficient generation of either photoluminescence, photoacoustic waves, or reactive oxygen species (ROS).[5-8] Such versatile and extraordinary properties have motivated researchers to explore the multifunctional Pdots for biological applications. Significant efforts have recently been focusedo ns ynthesizing new conjugated polymers, [9,10] developing functionalization strategies, [11,12] and designing signal transduction/amplification schemes. [13,14] The usefulness of Pdots in biological applications is strongly dependento nt he quality of the surfacef unctionalization to enables pecific recognition. Hence, many strategies have been implemented to modifyP dot surfaces with different functional molecules, for example, PEGylated lipids and amphiphilic polymers. [15,16] These functional molecules consist of hydrophobic components and hydrophilic segmentsw ith functional groups (e.g.,p olyethylene glycol,a mines, and carboxylic acids). During the Pdot formation, the hydrophobic segments are physically associated with polymer nanoparticles, while the hydrophilic chains extend partlyi nto the aqueous environment for further bioconjugation. Chiu's group developed ad irect functionalization strategy by modifying the side chain of the conjugated polymer.[17] Pdots formed from such functional polymers would directly have surface reactive groups for bioconjugation. In this strategy,the density of the side-chain functional groupss hould be well controlled to preventt he formationo farelatively loose structure, which can significantly affect nanoparticle stability and fluorescenceb rightness. However, challenges remain in the functionalization of Pdot surfaces for biomedical applications. Ideal semiconducting polymer nanoparticles ...