The major microbial process producing N₂O in estuarine ecosystems remains controversial. Combining the concentrations and isotopic compositions of N₂O, distributions and transcript levels of ammonia-oxidizing bacterial and archaeal amoA and denitrifier nirS genes, and in situ incubation estimates of nitrification rates and N₂O production rates, we clarified that ammonia-oxidizing bacteria contributed the major part in N₂O production in the upper Pearl River estuary despite their low abundance.