Newly emerging fluorescence imaging in the second near‐infrared window (NIR‐II, 1000–1700 nm) permits visualization of deep anatomical features with unprecedented spatial resolution due to its minimized autofluorescence, optical scattering and absorption of tissue, and increased applicable power at longer wavelengths than the traditional NIR (NIR‐I, 650–900 nm) fluorescence imaging. Compared with “always‐on” NIR‐II fluorescence probes, activatable NIR‐II fluorescence probes that can change their fluorescence signals by pathologic parameters stimuli, hold great promise for providing high target‐to‐background ratio, significantly improving specificity and sensitivity of early disease detection. Additionally, such probes provide an excellent opportunity to understand the underlying pathological mechanism at the molecular level in order to optimize therapeutic interventions. Here, the recent advances in the development of activatable NIR‐II fluorescence probes for bioimaging and biosensing are reviewed. The photochemical design mechanisms and biomedical applications of these probes are described in detail. Moreover, the present challenges to completely exhibit the potential of activatable NIR‐II fluorescence probes for optical theranostics in life science are also discussed. Finally, the future perspective is further analyzed.