To reveal the response mechanism of bacterial communities of biofilm to nitrate, this study took the submerged macrophyte Myriophyllum Verticillatum as the research object and explored the variations of bacterial community structure and related denitrification genes on the submerged macrophyte under four kinds of nitrate loading(2 mg/L, 8 mg/L, 20 mg/L and 40 mg/L), with the scanning electron microscope, 16S rRNA high-throughput sequencing and PCR amplification technology. The results showed that nitrate stimulated the growth of biofilm but reduced the bacterial α-diversity. The β_SOR diversity index showed that the bacterial community construction under nitrate loading was dominated by species replacements. Proteobacteria, Planctomycetes, Cyanobacteria and Bacteroidetes were the dominant phyla in biofilm. Compared with the control group, nitrate stimulated the growth of denitrifying bacteria(Rhodobacter, Acinetobacter and Bacillus)and the abundance of denitrifying genes(napG, nirS, cnorB, qnorB and nosZ). Network analysis showed that there were complex interactions among the microbes in biofilm. These results indicate that microbial denitrification plays an important role in the process of nitrate removal in wetland.