Polymethine cyanine dyes, as the most important class of organic near-infrared-II (NIR-II) fluorophores, recently received increasing attention due to their high molar extinction coefficients, intensive fluorescence brightness, and flexible wavelength tunability for fluorescent bioimaging applications. Very recently, remarkable advances have been made in the development of NIR-II polymethine fluorophores with improved optical performance, mainly including tunable fluorescence, improved brightness, improved water solubility and stability. In this review, we summarize the recent research advances in molecular tailoring design strategies of NIR-II polymethine fluorophores, and then emphasize the representative bioimaging and biosensing applications. The potential challenges and perspectives of NIR-II polymethine fluorophores in this emerging field are also discussed. This review may provide guidance and reference for further development of high-performance NIR-II polymethine fluorophores to boost their clinical translation in the future.