Multiphoton microscopy is an important biomedical imaging modality with great potential in the diagnosis of various diseases, due to high resolution, low damage, and long-term imaging of living organisms. Especially for the research on brain diseases, multiphoton microscopy can be used to observe neuron,blood vessels,tumors, and their interactions,which can further reveal the pathogenesis of brain diseases and direct the developement of diagnosis and treatment methods. This review introduces the basic principles and characteristics of multiphoton microscopy, and its applications in the study of various brain diseases, such as Alzheimer"s disease,stroke and tumor. We highlight recent achievements in brain diseases using multiphoton microscopy and give an outlook on the development of multiphoton microscopy that isexpected to play a greater role in the study of brain diseases.