Light‐based technique, including optical imaging and photoregulation, has become one of the most important tools for both fundamental research and clinical practice, such as cell signal sensing, cancer diagnosis, tissue engineering, drug delivery, visual regulation, neuromodulation, and disease treatment. In particular, low energy near‐infrared (NIR, 700–1700 nm) light possesses lower phototoxicity and higher tissue penetration depth in living systems as compared with ultraviolet/visible light, making it a promising tool for in vivo applications. Currently, the NIR light‐based imaging and photoregulation strategies have offered a possibility to real‐time sense and/or modulate specific cellular events in deep tissues with subcellular accuracy. Herein, the recent progress with respect to NIR light for monitoring and modulating the spatiotemporal dynamics of cell functions in living systems are summarized. In particular, the applications of NIR light‐based techniques in cancer theranostics, regenerative medicine, and neuroscience research are systematically introduced and discussed. In addition, the challenges and prospects for NIR light‐based cell sensing and regulating techniques are comprehensively discussed.