Estimation of surface energy fluxes using thermal remote sensing–based energy balance models (e.g., TSEB2T) involves the use of local micrometeorological input data of air temperature, wind speed, and incoming solar radiation, as well as vegetation cover and accurate land surface temperature (LST). The physically based Two-source Energy Balance with a Dual Temperature (TSEB2T) model separates soil and canopy temperature (Ts and Tc) to estimate surface energy fluxes including Rn, H, LE, and G. The estimation of Ts and Tc components for the TSEB2T model relies on the linear relationship between the composite land surface temperature and a vegetation index, namely NDVI. While canopy and soil temperatures are controlling variables in the TSEB2T model, they are influenced by the NDVI threshold values, where the uncertainties in their estimation can degrade the accuracy of surface energy flux estimation. Therefore, in this research effort, the effect of uncertainty in Ts and Tc estimation on surface energy fluxes will be examined by applying a Monte Carlo simulation on NDVI thresholds used to define canopy and soil temperatures. The spatial information used is available from multispectral imagery acquired by the AggieAir sUAS Program at Utah State University over vineyards near Lodi, California as part of the ARS-USDA Agricultural Research Service’s Grape Remote Sensing Atmospheric Profile and Evapotranspiration eXperiment (GRAPEX) project. The results indicate that LE is slightly sensitive to the uncertainty of NDVIs and NDVIc. The observed relative error of LE corresponding to NDVIs uncertainty was between -1% and 2%, while for NDVIc uncertainty, the relative error was between -2.2% and 1.2%. However, when the combined NDVIs and NDVIc uncertainties were used simultaneously, the domain of the observed relative error corresponding to the absolute values of |ΔLE| was between 0% and 4%.