Two-photon microscopy (TPM) plays an important role in the study of the changes of the two important components of neurovascular units (NVU) - neurons and blood vessels after ischemic stroke (IS). IS refers to sudden neurological dysfunction caused by focal cerebral ischemia, which is one of the leading causes of death and disability worldwide. TPM is a new and rapidly developing high-resolution real-time imaging technique used in vivo that has attracted increasing attention from scientists in the neuroscience field. Neurons and blood vessels are important components of neurovascular units, and they undergo great changes after IS to respond to and compensate for ischemic injury. Here, we introduce the characteristics and pre-imaging preparations of TPM, and review the common methods and latest progress of TPM in the neuronal and vascular research for injury and recovery of IS in recent years. With the review, we clearly recognized that the most important advantage of TPM in the study of ischemic stroke is the ability to perform chronic longitudinal imaging of different tissues at a high resolution in vivo. Finally, we discuss the limitations of TPM and the technological advances in recent years.