Ammonia nitrogen is a common pollutant in surface water, especially in agricultural areas, because the unreasonable application of nitrogen fertilizer will lead to the rapid increase of nitrogen concentration in surrounding water and maintain a high level. However, little is known about nitrogen loading on the decomposition of submerged macrophytes and N cycle genes in biofilms on plant debris. The alterations in epiphytic microbial abundances of nitrogen cycle-related genes and nutrient release were investigated in decomposition progress of Hydrilla verticillata, simulated in tanks with the concentrations of total nitrogen were 8 and 16 mg/L. The results show that there was no significant difference in the decomposition rates of Hydrilla verticillata residual between treatments with 8 and 16 mg/L total nitrogen. Compared with the control experiment, the dissolved oxygen concentrations and oxidation-reduction potential values decreased while the concentrations of carbon and total phosphorus rapidly increased in overlaying water in the early stage of plant decay. With prolonged decomposition time, the quality of overlying water was almost recovered back to the initial level (146 days). During the plant decomposition, the dissolved organic matter was composed of fulvic acid-like substances (including ultraviolet fulvic acid-like and visible fulvic acid-like) and protein-like substances (including tryptophan and tyrosine). Among five nitrogen cycle genes, changes in abundance of three genes were detected in biofilms on plant debris of two treatments. Redundancy analysis showed that the abundance of nitrogen cycle gene was less affected by the total nitrogen concentration of water, but was correlated with total organic carbon of plants, chemical oxygen demand, and dissolved oxygen concentration in overlaying water. Our results demonstrate that nitrogen loading did not affect the decomposition of plant debris but somewhat altered nitrogen cycle gene abundances in biofilm. However, the management of the aquatic vegetation (especially in the early stage of plant decomposition) and wastewater in the agriculture area still needs to be strengthened to reduce its impact on receiving water body.