This work reports the in vivo uptake and translocation of PNPs in the one-year grown terrestrial plant, Murraya exotica (M. exotica), as investigated by two-photon excitation and time-resolved (TPE-TR) optical imaging with a large field of view (FOV, 32 × 32 mm 2 ) in a noninvasive and real-time manner. The PNPs (⟨R h ⟩ = 12 ± 4.5 nm) synthesized from poly(styrene-co-maleic anhydride) (SMA) were Euluminescence labeled (λ L ≈ 617 nm). On exposing the roots of living M. exotica plants to the colloidal suspension of SMA PNPs at different concentrations, the spatiotemporal evolution of SMA PNPs along plant stems (60 mm in length) were monitored by TPE-TR imaging, which rendered rich information on the uptake and translocation of PNPs without any interference from the autofluorescence of the plant tissues. The TPE-TR imaging combined with the high-resolution anatomy revealed an intercellwall route in the lignified epidermis of M. exotica plants for SMA PNP uptake and translocation, as well as the similar accumulation kinetics at different positions along the plant stems. We modeled the accumulation kinetics with Gaussian distribution to account for the trapping probability of a SMA PNP by the lignified cell walls, allowing the statistical parameters, the average trapping time (t m ) and its variance (σ), to be derived for the quantification of the PNP accumulation in individual plants. The TPE-TR imaging and the analysis protocols established herein will be helpful in exploring the mechanism of plant-PNP interaction under physiological condition.