Nutrients (nitrogen and phosphorus) from agricultural non-point source (NPS) pollution have been increasingly recognized as a major contributor to the deterioration of water quality in recent years. The purpose of this article is to investigate the discrepancies in interception of nutrients in agricultural NPS pollution for eco-soil reactors using different filling schemes. Parallel eco-soil reactors of laboratory scale were created and filled with filter media, such as grit, zeolite, limestone, and gravel. Three filling schemes were adopted: increasing-sized filling (I-filling), decreasing-sized filling (D-filling), and blend-sized filling (B-filling). The systems were intermittent operations via simulated rainstorm runoff. The nutrient removal efficiency, biomass accumulation and vertical dissolved oxygen (DO) distribution were defined to assess the performance of eco-soil. The results showed that B-filling reactor presented an ideal DO for partial nitrification–denitrification across the eco-soil, and B-filling was the most stable in the change of bio-film accumulation trends with depth in the three fillings. Simultaneous and highest removals of NH4+-N (57.74–70.52%), total nitrogen (43.69–54.50%), and total phosphorus (42.50–55.00%) were obtained in the B-filling, demonstrating the efficiency of the blend filling schemes of eco-soil for oxygen transfer and biomass accumulation to cope with agricultural NPS pollution.