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.