The cyanine fluorophores, a kind of classic organic fluorophores, are famous for their high extinction coefficient, simple synthetic route, and relatively long absorption and emission wavelengths. Moreover, the excellent biocompatibility and low toxicity in biological samples make cyanine fluorophores show excellent application value in the biomedical field, especially in Near-Infrared II (NIR-II) sensing and imaging. In this review, we briefly outline the history, characteristics, and current state of development of cyanine fluorophores. In particular, we described the application of cyanine fluorophores in NIR-II sensing and imaging. We hope this review can help researchers grab the latest information in the fast-growing field of cyanine fluorophores for NIR-II sensing and imaging.