Fluorescence imaging has become an essential tool in biomedical research. However, non-invasive deep-tissue three-dimensional optical in vivo imaging with the high spatiotemporal resolution is challenging due to the interaction between photons and tissues. Beam shaping has been used to tailor microscopy techniques to enhance microscope performance. The near-infrared window (NIR) between 700 and 1,700 nm, generally emphasized as the NIR-II (1,000–1,700 nm) window, has been developed into a promising bio-optical solution chosen as the lower interaction effect in this spectrum, showing potential in basic biological research and clinical application. In this review, we summarize the existing methods to increase penetration depth and extensively describe biological microscopy techniques, NIR-II spectral windows, and fluorophores. Strategies to improve bioimaging performance and NIR-II imaging applications are introduced. Based on the current research achievements, we elucidate the main challenges and provide some recommendations and prospects for deep tissue penetration fluorescence for future biomedical applications.