Fluorescence imaging in the second near-infrared region (NIR-II, 1000-1700 nm) enables high-resolution visualization of deep-tissue biological architecture and physiopathological events, due to the reduced light absorption, scattering and tissue autofluorescence. Numerous versatile NIR-II fluorescent probes have been reported over the past decades. In this review, we first provide a detailed account of the advantages of fluorescence imaging in the NIR-II region. Following this, the classification, design and performance optimization strategies of NIR-II fluorescent probes are systematically discussed, along with a broad range of biomedical applications