Human activities are dramatically increasing estuarine nitrogen (N) loads, further altering N processes. However, relative importance of denitrification and anaerobic ammonium oxidation (anammox) in response to human activities intensity gradient remains poorly understood for estuaries. In this study, we used a 15N isotope tracer approach to characterize the variations in sediment denitrification and anammox rates and determined the crucial factors controlling the partitioning of N2 production and regulating N2O production across five subtropical estuaries in southeast China. Denitrification rates increased significantly from 8.82 ± 3.89 nmol N g−1h−1 (low human activity intensity) to 41.2 ± 11.5 nmol N g−1h−1 (high human activity intensity) across the studied estuaries. Anammox rates not varied significantly between low (2.37 ± 0.66 nmol N g−1h−1), moderate (3.96 ± 0.91 nmol N g−1h−1), and high (4.04 ± 1.09 nmol N g−1h−1) human activity intensity estuaries. Relative contribution of anammox to total N2 production (Ra) decreased toward estuary mouth within each estuary. The Ra was also significantly lower in the estuaries characterized by high N loads and low DO. N2O production rates were in a range of 0.192–1.92 nmol N2O g−1h−1 across the estuaries and controlled by water NO2–, salinity and TOC. DO, NH4+, and NO3– were the best predictors of the partitioning of N2 production between denitrification and anammox. The high human activities intensity increased NH4+ and NO3– loads and further enhanced denitrification, leading to the decrease in Ra and increase in N2O production. These findings suggest that low DO and high N loads estuaries can increase denitrification and N2O emissions, whereas not affect anammox substantially under the higher intensity of human activities.