Nitrate (NO3−) dissimilatory reduction processes (denitrification, anammox and dissimilatory NO3− reduction to ammonium (DNRA)) in estuarine and coastal ecosystems play a crucial role in regulating reactive nitrogen loadings. However, nitrate reduction process rates and relative proportions along the estuarine salinity gradient remain poorly understood. Here, denitrification, anammox and DNRA were explored simultaneously along a salinity gradient in Yangtze Estuary based on nitrogen isotope-tracing experiments. Measured denitrification, anammox and DNRA process rates were in the range of 2.33–28.21 nmol g−1 h−1, 0.43–1.87 nmol g−1 h−1 and 0.28–0.74 nmol g−1 h−1, respectively, with a large spatio-temporal variation. The changes in these nitrate reduction process rates were mainly affected by the TOC, TN, NH4+ and NOx− concentrations, rather than salinity and related functional gene abundance. Denitrification dominated the total NO3− reduction process (67.52 to 93.85%), while anammox (3.67 to 25.01%) and DNRA (2.48 to 11.21%) also played a substantially important role in nitrate reduction. The proportions of denitrification to gross nitrate reduction in high-salinity areas were generally lower than those in freshwater, but the opposite was true for DNRA. Overall, our study reported the simultaneous observation of nitrate dissimilatory reduction processes along the salinity gradient of the estuary and highlighted that changes in sediment environmental variables affected by human activities can alter the distribution patterns of NO3− reduction processes.