The applications of two-photon microscopy (TPM) on pre-clinical and clinical study of human cancer and diseases are reviewed in this paper. First, the principle of two-photon excitation (TPE) is introduced. The resulting advantages of TPM for imaging studies of animal models and human samples are then elaborated. Subsequently, the applications of TPM on various aspects of tumor studies, including tumor angiogenesis, invasion and metastasis, tumor microenvironment and metabolism are introduced. Furthermore, studies of TPM on clinical human skin biopsy and the development of two-photon microendoscopy are reviewed. Finally, potential future directions are discussed.Jun Liu’s current areas of expertise are optical microscopy, fluorescent imaging and pre-clinical imaging methods. His career focus is the application of advanced high resolution imaging methods on early detection and clinical treatment of human diseases. Throughout his early career, he has published over 14 peer-reviewed articles and received over 120 citations worldwide; he is also reviewer for various journals including PlosONE, Optics Letters. He is currently working in OptiMedic Technologies, Inc., a technology start-up company in Foshan, Guangdong, as a senior researcher and engineer to develop cutting edge uteroscope for early detection of cervical cancer.