Nitrate (NO3-) uptake and distribution are critical to plant life. Although the upstream regulation of NO3- uptake and downstream responses to NO3- in a variety of cells have been well studied, it is still not possible to directly visualize the spatial and temporal distribution of NO3- with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named NitraMeter3.0, for the quantitative visualization of NO3- distribution in Arabidopsis thaliana. This biosensor tracked the spatiotemporal distribution of NO3- along the primary root axis and disruptions by genetic mutation of transport (low NO3- uptake) and assimilation (high NO3- accumulation). The developed biosensor effectively monitors NO3- concentrations at the cellular level in real time and spatiotemporal changes during the plant life cycle.