<p>Agricultural non-point source pollution (AGNPS) currently affects 80% of global water bodies and 50% of land. Furthermore, rural rainwater runoff pollution is a common manifestation of AGNPS. Unlike domestic sewage, rural rainwater runoff typically exhibits high dissolved oxygen (DO) and low carbon-to-nitrogen (C/N) ratio characteristics. It primarily presents issues of total nitrogen (TN) exceeding standards, with nitrate nitrogen (NO₃-N) being the primary contributor. However, most of the current research results on the use of Constructed Rapid Infiltration Systems (CRIS) to treat rainwater runoff pollution have not achieved satisfactory results in terms of NO₃-N and TN removal, with some studies focusing only on NH₃-N removal while neglecting NO₃-N. Addressing these limitations, this study employed artificially simulated rainwater runoff as influent to conduct comparative experiments to evaluate the purification efficiency of four different filter media combinations within an iron-based autotrophic denitrification CRIS. The evaluated parameters were COD, NH₃-N, NO₃-N, TN, and TP, while changes in pH were simultaneously monitored. Additionally, at the end of the experiment, biodiversity testing was conducted on the filter media in the CRIS of the four schemes. After 49 days of operation, the results demonstrated that the iron-based autotrophic denitrification CRIS equipped with filter media combination B (zeolite: laterite-sand mixture: sponge iron: activated carbon = 1:1:1:2) achieved optimal performance. Its average removal rates for COD, TN, NO₃-N, NH₃-N, and TP reached 68.51%, 74.43%, 80.97%, 92.03%, and 98.74%, respectively. The microbial community analysis also substantiates that the filter media in Scheme B possessed superior denitrification capabilities. The results of this study provide a simple and effective new technology for controlling rural rainwater runoff pollution.</p>