Description: Nitrous oxide (N2O) is a greenhouse gas, with a global warming potential 298 times that of carbon dioxide. Estuaries can be sources of N2O, but their emission estimates have significant uncertainties due to limited data availability and high spatiotemporal variability. We investigated the spatial and seasonal variability of dissolved N2O and its emissions along the Elbe Estuary (Germany), a well-mixed temperate estuary with high nutrient loading from agriculture. During nine research cruises performed between 2017 and 2022, we measured dissolved N2O concentrations, as well as dissolved nutrient and oxygen concentrations along the estuary, and calculated N2O saturations, flux densities, and emissions. We found that the estuary was a year-round source of N2O, with the highest emissions in winter when dissolved inorganic nitrogen (DIN) loads and wind speeds are high. However, in spring and summer, N2O saturations and emissions did not decrease alongside lower riverine nitrogen loads, suggesting that estuarine in situ N2O production is an important source of N2O. We identified two hotspot areas of N2O production: the Port of Hamburg, a major port region, and the mesohaline estuary near the maximum turbidity zone (MTZ). N2O production was fueled by the decomposition of riverine organic matter in the Hamburg Port and by marine organic matter in the MTZ. A comparison with previous measurements in the Elbe Estuary revealed that N2O saturation did not decrease alongside the decrease in DIN concentrations after a significant improvement of water quality in the 1990s that allowed for phytoplankton growth to re-establish in the river and estuary. The overarching control of phytoplankton growth on organic matter and, subsequently, on N2O production highlights the fact that eutrophication and elevated agricultural nutrient input can increase N2O emissions in estuaries.