Excess nitrogen (N) in lakes may lead to eutrophication and many attendant environmental problems such as water quality decline and loss of aquatic biodiversity. Denitrification in lake sediments can alleviate the effects of eutrophication through removal of N to the atmosphere as N2O and N2. However, N2O contributes to the greenhouse effect and global warming. In this study, we measured three denitrification parameters (i.e., potential denitrification rate, unamended denitrification rate and net N2O production rate) in surface sediments which were collected from 7 sub-lakes of the Lake Donghu, one of the most eutrophic lakes in China. The results showed that a range of water quality and sediment characteristics (e.g., total N and total phosphorus) varied significantly among sub-lakes. The unamended denitrification rate varied between 0.51 and 26.0 ng N g−1 h−1, while the N2O production rate ranged from less than 0 to 1.68 ng N g−1 h−1. However, there was no significant difference among the sub-lakes in these denitrification parameters. The unamended denitrification rate was positively related to the water NO3− concentration and sediment moisture and bulk density. The findings of the present study suggest that sediments in eutrophic lakes can remove large quantities of N through denitrification and may become a significant source of N2O if the N input is maintained or to increase.