Phototrophic biofilms are easy to grow at sediment/soil–water interfaces (SWIs) in shallow aquatic ecosystems and greatly impact nutrient biogeochemical cycles. However, the pathways by which they contribute to nitrogen interception and interact with sediment/soil remains largely unknown. Here, we conducted a field investigation in paddy fields in various regions of China and found that nitrogen immobilized in biofilm biomass significantly positively correlated with soil organic carbon (SOC) content. A microcosm experiment showed that this was due to increased bacterial and algal diversity, biomass accumulation, and inorganic nitrogen assimilation at high SOC, especially high dissolved organic carbon (DOC) levels. The metatranscriptomics results further verified that many KO groups of PSII, PSI, AP, and PC in antenna proteins and glutamate synthesis were distinctly expressed at elevated SOC and DOC levels. Our results elucidated the effects and possible pathways of how SOC enrichment triggers photosynthesis and nitrogen immobilization by phototrophic biofilms. The results will provide meaningful information for in situ nitrogen interception by using phototrophic biofilms at the SWI in human-made wetlands to change internal nitrogen cycling.