Brain interstitial system (ISS) is a nanoscale network of continuously connected tubes and sheets surrounding each neural cell in the central nervous system. ISS usually accounts for ∼20% of the brain volume, far more than the cerebral blood vessels, which account for 3%. The neuronal function, signaling pathways, and drug delivery are all closely related to the microenvironment provided by ISS. The objective of this paper is to give the readers a clear outline of detection, anatomy, function, and applications of ISS. This review describes the techniques propelling the exploration for ISS in chronological order, physiological function and pathological dysfunction of ISS, and strategies for drug delivery based on ISS. Biophysical features are the focus of ISS research, in which the diffusion characteristics have dominated. The various techniques that explore ISS take advantage of this feature. ISS provides an essential microenvironment for the health of cells and brain homeostasis, which plays an important functional role in brain health and disease. Direct intracranial administration allows the diffusion of drugs directly through ISS to successfully bypass the blood–brain barrier that prevents most drugs from reaching the brain. With the deepening of understanding of the brain ISS, the new research model that takes into account brain cells, cerebral vessels, and ISS will provide a new perspective and direction for understanding, utilizing, and protecting the brain.