Abstract: In recent years, ecological ditch technology has received attention in the treatment of agricultural non-point source (NPS) pollution, but the technology still suffers problems such as low pollutant removal efficiency, which needs to optimize its structure and improve the treatment efficiency of typical pollutants. In view of the characteristics of typical NPS pollutants, three ecological ditches were constructed with different substrates (limestone, volcanic rock, zeolite and mafic rock as filler substrates in ED₁ and ED₂, and zeolite, ceramic granule and slag in ED₃) and plant configurations (Acorus calamus, Pontederia cordata and Hydrocotyle vulgaris were planted in ED₁ and ED₃, and Acorus calamus, Canna indica and Hydrocotyle vulgaris were planted in ED₂) to explore the effects on the removal of typical NPS pollutants, such as ammonia nitrogen (\mathrmNH_4^+ -N), total nitrogen(TN), and total phosphorus(TP). The potential mechanisms between pollutant removal performance and microorganisms were also explored through high-throughput sequencing. The results showed that limestone, volcanic rock, zeolite and mafic rock as filler substrate with planting of Acorus calamus, Canna indica and Hydrocotyle vulgaris (ED₂) was the preferred form of grouping, which could remove up to 100%, 86.89% and 87.42% of \mathrmNH_4^+ -N, TN and TP, respectively, and ED₂ was the best for removing NPS pollutants. The concentration of pollutants in the influent, hydraulic retention time (HRT), and water level had a significant impact on the effectiveness of pollutant removal in ecological ditches. The highest removal rates of \mathrmNH_4^+ -N, TN and TP pollutants were obtained under the conditions of the HRT of 12 h, mean water level (6 cm) and high concentration of the pollutants (\mathrmNH_4^+ -N=15 mg/L, TN=40 mg/L, and TP=1.2 mg/L), and the removal rates of \mathrmNH_4^+ -N, TN and TP could reach 99%-100%, 41%-50%, and 81%-88%, respectively. Microbial diversity analysis showed that Proteobacteria, Actinobacteriota and Chloroflexi were the main dominant phyla in the ecological ditch, and these phyla played a key role in the removal of nitrogen and phosphorus. The relative abundance of species of dominant phyla was the highest in ED₂ after the operation, among which Canna indica had a higher abundance of specific microbial species with the highest diversity of microbial community in ED₂, the highest species abundance of Hydrocotyle vulgaris was observed in ED₂, the abundance of Proteobacteria in ED₂ was increased from 25.49% to 45.11%, and the change in microbial community abundance was positively correlated with pollutant removal efficiency. The results of this study can provide a reference for the design and configuration optimization of ecological ditches.