Understanding the interaction mechanisms of engineered nanomaterials (ENMs) with plant membranes is crucial for their effective use in various applications. While passive transport of smaller ENMs is well-documented, the mechanisms underlying active transport of larger ENMs remain poorly understood. This study systematically investigates the active transport and subcellular distribution of ENMs (100–1000 nm) within protoplasts using optical ratiometric silica pH sensors for localization. Highly monodispersed ratiometric pH sensors, based on silica particles modified with fluorescein-5-isothiocyanate (FITC) and cyanine3 NHS ester (CY3) dyes, were employed to elucidate internalization mechanisms. Protoplasts from Nicotiana tabacum L. leaves successfully internalized the sensors. 3D segmentation of protoplasts revealed distinct pH gradients, indicating vacuole accumulation. Colocalization analysis and cellular compartments staining further confirmed sensor distribution. High-throughput imaging flow cytometry showed efficient internalization rates, which decreased after cell wall regeneration. Notably, inhibition experiments with the salicylic acid (SA) and Tyrphostin A23 (TyrA23) inhibitors confirmed clathrin-mediated endocytosis in particle uptake. This study establishes rational design principles for controlling active ENM uptake and subcellular localization via optical pH sensing in protoplasts. The findings enhance our understanding of plant cell trafficking mechanisms and hold promise for targeted delivery and applications in plant biology research.