Two-photon microscopy, with its exceptional capabilities in deep tissue penetration ability, low phototoxicity, high spatial resolution, anti-light scattering properties, and long working distance, has become an ideal tool for imaging living tissues, deep structures, and optically cleared thick-tissue samples, and is widely used in the field of biomedical research. However, the application of this technique in acupuncture research is still in its early stage. Given the great potential of two-photon microscopy imaging in acupuncture research, this article systematically explains the principles and technical advantages of two-photon microscopy, and reviews its latest application progress in the field of acupuncture and moxibustion. At the same time, by integrating second harmonic generation imaging, optogenetics, gene editing, and tissue clearing techniques, innovative strategies tailored for acupuncture and moxibustion research are proposed. Concrete suggestions are also provided for achieving high-quality imaging with two-photon microscopy. These efforts aim to facilitate the advanced application of this technology in acupuncture and moxibustion research, enable real-time dynamic in vivo observation of acupuncture effects, and provide robust technical support for elucidating the underlying mechanisms of acupuncture and moxibustion.