Optical methods can show different chemical components in tissues and are non-invasive, non-destructive, and non-ionizing. Near-infrared II light distinguishes itself among numerous light wavelengths by virtue of its unique advantages and has become a more preferred choice when selecting light wavelengths. Its notable advantages include being able to penetrate deep into biological tissues with greater efficacy and effectively reducing the impact of tissue scattering and absorption phenomena. Utilizing fluorophores in the near-infrared II window generates superior diagnostic spatial resolution, additional signal-to-background ratio, and greater depth of tissue optical imaging. Organic small molecule fluorophores, as a new generation of fluorophores with potential for clinical application, have made remarkable progress in biomedical imaging, drug delivery, and cancer therapy in near-infrared II and have become a research hotspot. This review is concentrated on the advancements made recently by different near-infrared II emitting organic small molecule fluorophores in the domains of molecular structure design, methods to improve biocompatibility, water solubility, targeted transport, bioimaging, phototherapy, near-infrared II emitting organic small molecule fluorophores prospects and challenges. We hope to open a path for the progress of future near-infrared II emitting organic small molecule fluorescence technology and accelerate its application in clinical medicine.