Aquaculture supplies high-quality and healthy proteins. With the increasing human demand for aquaculture production, intensive pond aquaculture developed rapidly and results in environmental deterioration. To solve this problem, the eco-substrate (ES), which is the biofilm carrier, has been utilized in aquaculture ponds. Studying the ecological mechanisms of ES from the perspective of the ecosystem may be conducive to the sustainable development of aquaculture. In this study, it was evaluated how ES makes a difference to the trophic structure, energy flow, and system characteristics of two different aquaculture pond ecosystems via the ecopath model. Three aquaculture ponds with ES were designed as the treatment ecosystem and three aquaculture ponds without ES were designed as the control ecosystem. There were 13 and 14 functional groups in the control and treatment ecosystems, respectively. The results showed that (1) the macrozooplankton and microzooplankton showed strong effects on the ecosystem in the keystoneness index; (2) energy transfer pathways in the treatment system with ES increased by 26.23% compared to the control system; (3) the ES improved the utilization rate of detritus, which was 14.91% higher than that of the control ecosystem; (4) the material and energy flow index and network information characteristics demonstrated the ES enhanced the complexity and stability of the treatment system. To improve the energy utilization efficiency, filter feeders can be introduced to ES ponds. Overall, the ES can alter the trophic structure, improve the energy utilization efficiency, and enhance the stability and maturity of aquaculture ecosystems, representing a sustainable practice. Considering the total area of aquaculture ponds on the earth reaching more than 5 million hectares, the application prospect of ES is broad.