Perivascular pericytes are key regulators of the blood–brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, pericyte identification remains challenging. A previous study reported that the far-red fluorophore TO-PRO-3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO-PRO-3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO-PRO-3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron-glial antigen 2 (NG2) and platelet-derived growth factor receptor beta antigen (PDGFrβ) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO-PRO-3 signal enabled quantification of pericytes density and morphometry; likewise, TO-PRO-3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO-PRO-3 stained cells expressed the contractile protein α–SMA, indicative of their ability to control the capillary diameter. Uptake of TO-PRO-3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein-mediated active transport sustained dye incorporation. We conclude that TO-PRO-3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature.