This article describes the design and synthesis of quinoxaline-based semiconducting polymer dots (Pdots) that exhibit near-infrared fluorescence, ultrahigh brightness, large Stokes shifts, and excellent cellular targeting capability. We also introduced fluorine atoms and long alkyl chains into polymer backbones and systematically investigated their effect on the fluorescence quantum yields of Pdots. These new series of quinoxaline-based Pdots have a fluorescence quantum yield as high as 47% with a Stokes shift larger than 150 nm. Single-particle analysis reveals that the average per-particle brightness of the Pdots is at least 6 times higher than that of the commercially available quantum dots. We further demonstrated the use of this new class of quinoxaline-based Pdots for effective and specific cellular and subcellular labeling without any noticeable nonspecific binding. Moreover, the cytotoxicity of Pdots were evaluated on HeLa cells and zebrafish embryos to demonstrate their great biocompatibility. By taking advantage of their extreme brightness and minimal cytotoxicity, we performed, for the first time, in vivo microangiography imaging on living zebrafish embryos using Pdots. These quinoxaline-based NIR-fluorescent Pdots are anticipated to find broad use in a variety of in vitro and in vivo biological research.