Global nitrogen (N) enrichment has resulted in increased nitrous oxide (N2O) emission that greatly contributes to climate change and stratospheric ozone destruction, but little is known about the N2O emissions from urban river networks receiving anthropogenic N inputs. We examined N2O saturation and emission in the Shanghai city river network, covering 6300 km2, over 27 months. The overall mean saturation and emission from 87 locations was 770% and 1.91 mg N2O-N m−2 d−1, respectively. Nitrous oxide (N2O) saturation did not exhibit a clear seasonality, but the temporal pattern was co-regulated by both water temperature and N loadings. Rivers draining through urban and suburban areas receiving more sewage N inputs had higher N2O saturation and emission than those in rural areas. Regression analysis indicated that water ammonium (NH4+) and dissolved oxygen (DO) level had great control on N2O production and were better predictors of N2O emission in urban watershed. About 0.29 Gg N2O-N yr−1 N2O was emitted from the Shanghai river network annually, which was about 131% of IPCC's prediction using default emission values. Given the rapid progress of global urbanization, more study efforts, particularly on nitrification and its N2O yielding, are needed to better quantify the role of urban rivers in global riverine N2O emission.