AbstractThe near‐infrared window between 1000 and 1700 nm, commonly termed the “second near‐infrared (NIR‐II) window,” has quickly emerged as a highly attractive optical region for biological imaging. In contrast to conventional imaging in the visible region between 400 and 700 nm, as well as in the first NIR (NIR‐I) window between 700 and 900 nm, NIR‐II biological imaging offers numerous merits, including higher spatial resolution, deeper penetration depth, and lower optical absorption and scattering from biological substrates with minimal tissue autofluorescence. Noninvasive imaging techniques, specifically NIR‐II fluorescence and photoacoustic (PA) imaging, have embodied the attractiveness of NIR‐II optical imaging, with several NIR‐II contrast agents demonstrating superior performance to the clinically approved NIR‐I agents. Consequently, NIR‐II biological imaging has been increasingly explored due to its tremendous potential for preclinical studies and clinical utility. Herein, the progress of optical imaging in the NIR‐II window is reported. Starting with highlighting the importance of biological imaging in the NIR‐II spectral region, the emergence and latest development of various NIR‐II fluorescence and PA imaging probes and their applications are then discussed. Perspectives on the promises and challenges facing this nascent yet exciting field are then given.