Biological nitrogen fixation (BNF) is a crucial process that provides bioavailable nitrogen and supports primary production in freshwater lake ecosystems. However, the characteristics of diazotrophic community and nitrogenase activity in freshwater lake sediments remain poorly understood. Here, we investigated the diazotrophic communities and nitrogenase activities in the sediments of three large river-connected freshwater lakes in eastern China using 15 N-isotope tracing and nifH sequencing. The sediments in these lakes contained diverse nitrogenase genes that were phylogenetically grouped into Clusters I and III. The diazotrophic communities in the sediments were dominated by stochastic processes in Hongze Lake and Taihu Lake, which had heterogeneous habitats and shallower water depths, while in Poyang Lake, which had deeper water and a shorter hydraulic retention time, the assembly of the diazotrophic community in the sediments was dominated by homogeneous selection processes. Temperature and water depth were also found the key environmental factors affecting the sediment diazotrophic communities. Sediment nitrogenase activities varied in the three lakes and within distinct regions of an individual lake, ranging from 0 to 14.58 nmol/(kg·hr). Nitrogenase activity was significantly correlated with ferric iron, total phosphorus, and organic matter contents. Our results suggested that freshwater lake sediment contain high diversity of nitrogen-fixing microorganisms with potential metabolic diversity, and the community assembly patterns and nitrogenase activities varied with the lake habitat.