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[Display omitted] • A selection strategy for base-period images of spatio-temporal fusion was proposed. • The spatio-temporal inconsistency between field samples and images was reduced. • The estimation accuracy of grassland aboveground biomass was improved. The accurate estimation of the aboveground biomass (AGB) is crucial for the sustainable utilization and management of grassland resources. Spatio-temporal inconsistencies between field samples and satellite images are major source of error in the estimation of grassland AGB. To solve this problem, this study selected the Three Rivers Headwater Region as the study area and proposed a selection strategy for base-period images of spatio-temporal fusion that was suitable for use at a large spatial scale in which cloud-free images are difficult to obtain. The spatial and temporal adaptive reflectance fusion model (STARFM) based on the selection strategy was used to generate a synthetic Normalized Difference Vegetation Index (NDVI) dataset with high spatial-temporal resolution by using the maximum value composite of GF-1 NDVI and MODIS NDVI to enhance the spatial-temporal quality of the images for field-scale application. Three estimation models for grassland AGB were then constructed by the random forest algorithm using synthetic NDVI, MODIS NDVI, GF-1 NDVI respectively, together with ancillary data. Following this, the estimation model with the highest accuracy was used to generate a 16-m eight-day time-series AGB in the growing season. The results showed: (1) The synthetic NDVI was correlated closely with the observed GF-1 NDVI, with an average R of 0.825 and a RMSE of 0.087. The temporal trend of the synthetic NDVI for each grassland type was highly consistent with that of the MODIS NDVI in the growing season with a correlation higher than 0.9. (2) The synthetic NDVI reduced the spatial difference between field samples and images to 16-fold, and the temporal difference was controlled to within four days under ideal conditions. (3) The synthetic NDVI improved the estimation accuracy of grassland AGB by about 15.9% and 19.7% (R2), and 13.7% and 17.5% (RMSE) relative to MODIS NDVI and GF-1 NDVI, respectively. (4) The time-series AGB revealed accurately the spatial distribution of and seasonal temporal variations in the grassland biomass. The results of this study may serve as scientific guidance for timely monitoring of grassland conditions and precise management of grassland resources in China. [ABSTRACT FROM AUTHOR]