This review discusses the recent advancements in semiconducting polymer nanoparticles, known as polymer dots (Pdots), which have gained significant attention due to their outstanding characteristics as fluorescent probes. Pdots, primarily consisting of π-conjugated polymers, exhibit high brightness, fast emission rates, excellent photostability, nonblinking, and nontoxicity. The review highlights the photophysical properties of Pdots, their surface functionalization, and biomolecular conjugation, as well as their applications in cellular labeling, in vivo imaging, single-particle tracking, biosensing, and drug delivery. It also evaluates the relationship between physical properties and performance, and addresses the current challenges and future directions in the field. Pdots have shown superior performance compared to conventional fluorescent dyes and inorganic quantum dots, making them promising probes for advanced imaging techniques and high-throughput assays in biology and medicine.This review discusses the recent advancements in semiconducting polymer nanoparticles, known as polymer dots (Pdots), which have gained significant attention due to their outstanding characteristics as fluorescent probes. Pdots, primarily consisting of π-conjugated polymers, exhibit high brightness, fast emission rates, excellent photostability, nonblinking, and nontoxicity. The review highlights the photophysical properties of Pdots, their surface functionalization, and biomolecular conjugation, as well as their applications in cellular labeling, in vivo imaging, single-particle tracking, biosensing, and drug delivery. It also evaluates the relationship between physical properties and performance, and addresses the current challenges and future directions in the field. Pdots have shown superior performance compared to conventional fluorescent dyes and inorganic quantum dots, making them promising probes for advanced imaging techniques and high-throughput assays in biology and medicine.