Nitrogen fixation is a biogeochemical process that plays a vital role in river ecology and can occur in sediments and soils. The present investigation was carried out over 50 km to reach the Padma River of Bangladesh, downstream of the confluence of the Ganges and Brahmaputra rivers. The study area is highly dynamic, with various land-use–land-cover (LULC) types such as cropland (CL), natural vegetation (NV), dry, bare land (DBL) and land with water (LW). A field study was carried out during the low flow (dry/winter) season to measure the nitrogen fixation rate (NFR) using the acetylene reduction method for each type of LULC. Linear regression using a mixed-modelling approach showed that NFR was highly related to sediment bulk density (SBD) and moisture across all the LULC types. Sentinel-2 data were then used to develop relationships between band 11 and SBD. Later, NFR was upscaled in different seasons based on the random forest algorithm, which relied solely on Sentinel-2 products. The results showed that seasonal changes in the surface area and number of LULC types could alter NFR in the study reach. This satellite-based estimation of the spatial and temporal distribution of NFR can more precisely demonstrate the role of NFR as an ecosystem service. Moreover, to better estimate the nitrogen budget of large rivers, consideration of NFR is essential, especially in the case of large lowland rivers where seasonal inundation occurs frequently.