To study nitrogen removal by Ulva lactuca and its effects on the structure of the bacterial community and abundance of nitrogen cycle functional genes in the ‘sea cucumber–shrimp–crab–fish’ integrated multi-trophic aquaculture (IMTA) system, we analysed bacterial community composition by Illumina MiSeq high-throughput sequencing and nitrogen cycling functional gene levels by real-time quantitative PCR in water and sediment with (Y) and without the presence of U. lactuca (N). Diversity and abundance indices, including Sobs, Shannon, Ace, and Chao1, were higher in Group Y water and sediment. In the water, the predominant phyla were Proteobacteria in Group Y and Cyanobacteria in Group N in September. Flavobacteriaceae and Rhodobacteraceae were the dominant families in Group Y and Group N in October and November, respectively. In the sediment, Bacillaceae was the dominant family in Group Y throughout the experimental period, whereas Desulfocapsaceae was the dominant family in Group N in October and November. Regarding nitrogen cycle functional genes, millions of Amx, nosZ, nxrB, and nrfA genes were detected in both water and sediment. The abundances of nifH, amoA, nxrB, norB, and nrfA genes were higher in Group Y than those in Group N, which can improve ammonia removal without nitrogen loss. In this study, a potential novel approach was proposed to enhance the development of sustainable aquaculture, promote the nitrogen cycle, and remediate and optimise the water environment by cultivating bacteria and algae, according to the effects of U. lactuca on water quality, bacterial communities, and abundance of functional nitrogen cycling genes in IMTA systems.