Compartmentalized nitric oxide (NO) production drives critical signaling pathways in cells, yet there are no methods to quantitatively image NO with sub-cellular spatial resolution in living systems. Here, we introduce a new DNA-based fluorescent reporter technology that maps NO with sub-cellular resolution in live cells. It combines small molecule NO detection chemistry with the sub-cellular targetability of DNA based scaffold to provide quantifiable NO maps using ratiometric imaging. We could thereby map the activity of Nitric Oxide Synthase 3 (NOS3) which resides at the plasma membrane and the trans-Golgi network. We find that despite its lower abundance, the pool of NOS3 at the plasma membrane is seven-fold more active than Golgi associated NOS3. The ability to quantitatively map NO dynamics with sub-cellular resolution provides the potential to discover selective regulators of distinct NOS3 populations.