Wetlands are a critical component of the landscape for climate mitigation, adaptation, biodiversity, and human health and prosperity. Keeping an eye on wetland vegetation is crucial due to it playing a major role in the planet’s carbon cycle and ecosystem management. By measuring the chlorophyll fluorescence (ChF) emitted by plants, we can get a precise understanding of the current state and photosynthetic activity. In this study, we applied the Extreme Gradient Boost (XGBoost) algorithm to map ChF in the Biebrza Valley, which has a unique ecosystem in Europe for peatlands, as well as highly diversified flora and fauna. Our results revealed the advantages of using a set of classifiers derived from EO Sentinel-2 (S-2) satellite image mosaics to accurately map the spatio-temporal distribution of ChF in a terrestrial landscape. The validation proved that the XGBoost algorithm is quite accurate in estimating ChF with a good determination of 0.71 and least bias of 0.012. The precision of chlorophyll fluorescence measurements is reliant upon determining the optimal S-2 satellite overpass time, which is influenced by the developmental stage of the plants at various points during the growing season. Finally, the model performance results indicated that biophysical factors are characterized by greenness- and leaf-pigment-related spectral indices. However, utilizing vegetation indices based on extended periods of remote sensing data that better capture land phenology features can improve the accuracy of mapping chlorophyll fluorescence.