Organic polymethine fluorophores with emission in the NIR-II window (1000-1700 nm) are receiving enormous attention in biomolecular medicine and bioimaging, attributed to the high absorption coefficients, bright NIR-II emission, excellent biocompatibility, and molecule adjustability. Recently, researchers have devoted significant effort to designing and improving NIR-II polymethine fluorophores and making notable progress in the NIR-II fluorescence imaging performance. This review summarizes recent developments in the molecular engineering design mentality of polymethine fluorophores and probes and highlights their extensive bioimaging and biosensing applications. Furthermore, we elucidate the potential challenges and perspectives of these NIR-II polymethine dyes in chemical and biomedicine imaging, which may stimulate the further development of high-performance NIR-II polymethine dye contrast agents for their future clinical applications.