Near-infrared (NIR) bioimaging has emerged as a transformative technology in biomedical research. Among many fluorescent probes that are suitable for NIR imaging studies, two-photon absorption (TPA) ones represent a particularly promising category, because TPA fluorescent probes can overcome the inherent limitations of one-photon absorption (OPA) counterparts. By leveraging the unique properties of two-photon absorption, TPA fluorescent probes achieve superior tissue penetration, significantly reduced photodamage, and enhanced spatial resolution. This perspective article delves into the fundamental principles, design strategies, and representative TPA probes for various imaging applications. In particular, a number of molecular fluorescent probes, ranging from organic, inorganic, and COF/MOF-based systems are highlighted to showcase the vast scope of possible TPA probe design and application scenarios. In addition, the employment of stimulated TPA probes that are responsive to different external factors, including pH, redox species, enzymes, and hypoxia, is also discussed. In the end, the future perspectives for the continuous advancement of TPA fluorescent probes in the NIR bioimaging field are presented. For instance, it is essential to transition from cellular to in vivo imaging studies to obtain more physiologically relevant insights. Additionally, the development of "dual-function" TPA probes for both disease diagnosis and therapeutic treatment is particularly promising.