Urban trees contribute to urban well-being but face challenging environments that can reduce their lifespan and increase young tree mortality. Although many studies have used remote sensing data to monitor the functional status of trees in rural areas, few have done so in urban areas to assess the health or estimate the biomass of large green areas. This study assessed the suitability of using Sentinel-2 images to characterize two urban tree functional traits—leaf chlorophyll content (Cab) and leaf area density (LAD)—in isolated trees and tree rows. Simulated Sentinel-2 images were generated using the DART radiative transfer model, considering 16 tree-endogenous and 14 tree-exogenous parameters, with 15 vegetation indices (VIs) analyzed. Sensitivity analysis was performed in four contrasting urban environments using local climate zone taxonomy. The accuracy of the simulated images was validated with real Sentinel-2 images, field measurements, and ancillary data collected for four tree species in Rennes, France. The results showed that the tree parameters significantly influenced Sentinel-2 spectral bands, with NGBDI and OSAVI VIs being most sensitive to Cab and LAD. The model showed high accuracy, with a mean RMSE of 0.016 for key spectral bands. The results also highlighted the importance of considering ancillary data to capture specific urban characteristics.