Carbon dioxide (CO2) is a more powerful greenhouse gas due to its enhanced instantaneous radiative forcing. Since agricultural ecological ditches (eco-ditches) controlling agricultural non-point source pollution are potential CO2 hotspots, carbon capture technologies must be deployed immediately to sequester carbon and combat climate change. Zero-waste biochar, a negative-carbon technology, remains contentious regarding C sequestration. Iron (Fe) enhances long-term organic carbon (OC) preservation, but the cascading effects of Fe-biochar interactions on the promotion of C accumulation are unclear in eco-ditches. In this paper, we fill these research gaps and employ sponge iron (s-ZVI) and biochar coupling (Fe-C) to enhance the iron gate C protection in eco-ditches. The results reveal that Myriophyllum aquaticum(M.A.)-derived biochar prepared by low-temperature pyrolysis (Biochar_M.A.) enjoys the highest FI, the lowest SUVA254 and SUVA280 and moderate dissolution stability. The coupling of s-ZVI and environmentally compatible Biochar_M.A. achieve a triple benefit situation of C sequestration, C sink, and pollutant removal. Overall carbon gain is jointly determined by abiotic controls and biotic controls. The carbon burial effects of biochar-amended eco-ditches are mainly controlled by the trade-offs between Fe-bound OC mineralization via the biochar-induced prime effect and preservation of OC derived from microbial biomass. The unique Fe-C boosts the rusty sink and yields a negative priming as a result of the dominance of Fe-OC. The upregulated β-glucosidase activity and new-formed Fe-OC indirectly and directly contribute to overall C gain in Fe-C-filled eco-ditches, respectively. These findings provide novel insights into the coupled biotic-abiotic contribution mechanisms underlying the iron gate and reverse enzyme latch phenomenon and advance more robust and prospective theoretical knowledge of C dynamics that are not restricted to eco-ditches, soil, constructed wetlands C storage.