AbstractNear‐infrared (NIR) fluorescent materials are considered to be the most promising labeling reagents for sensitive determination and biological imaging due to the advantages of lower background noise, deeper penetrating capacity, and less destructive effects on the biomatrix over those of UV and visible fluorophores. In the past decade, advances in biomedical fluorescence imaging in the NIR region have focused on the traditional NIR window (NIR‐I; λ=700–900 nm), and have recently been extended to the second NIR window (NIR‐II; λ=1000–1700 nm). In vivo NIR‐II fluorescence imaging outperforms imaging in the NIR‐I window as a result of further reduced absorption, tissue autofluorescence, and scattering. In this review, the applications of four types of NIR‐II fluorescent materials, organic fluorophores, quantum dots, rare‐earth compounds, and single‐walled carbon nanotubes, are summarized and future trends are discussed. Some methods to enhance the NIR‐II fluorescence quantum yield are also proposed.