Standard histopathology is well accepted as the gold standard for the diagnosis a wide range of diseases. Despite continuing advances in tissue staining automation, typical histological processing such as formalin-fixed paraffin-embedded are also labour- and time-intensive for treatment decisions in intraoperative histopathologic diagnosis. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), can be a versatile tool that enables label-free mapping of endogenous fluorophores within a fresh specimen, which provides pathology-like images with cellular and subcellular details. Here, we describe the use of label-free MPM for visualizing rat and human ex vivo brain tissue without tissue fixation, processing, and staining. Moreover, MPM is able to identify 6 types of cells in rat cerebrum and cerebellum, including cortical neurons, glia cells, Purkinje cells, pyramidal neurons and granule neurons in hippocampus, as well as epithelial cells in lateral ventricle. In addition, we further demonstrate that MPM can provide definitive pathological features in cerebral ischemia and focal cortical dysplasia (FCD) for assisting pathologic diagnosis. Our work establishes the methodology and augments the diagnostic accuracy of traditional frozen section histopathology. With the development of the miniature two-photon microscope, MPM will show more potential as a practical clinical tool for providing intraoperative reference image guidance of resection in neurosurgery.