To investigate the relationship between the relative abundance of fish environmental DNA (eDNA) sequence and number of individual in a pond model. In September 2022, 100 Myxocyprinus asiaticus , 800 Leiocassis longirostris , 1 000 Megalobrama pellegrini and 2 500 Spinibarbus sinensis were temporarily cultured in a pond with dimensions of 200 m in length, 100 m in width, and 2 m in depth. After 10 days, 15 sampling sites were set up in this pond. The relationship between number of individuals of the above four fish species and the relative abundance of eDNA sequences were detected through a series of standardized analytical processes, including water sample collection, filtration, eDNA extraction, PCR amplification, high-throughput sequencing and biosignature analysis using the fish mitochondrial gene 12S gene as a marker. The results showed: 1) The relative abundance of M. asiaticus sequence was the lowest (3 970 sequences), and that of S. sinensis was the highest (687 895 sequences). 2) The relative abundance of eDNA sequence for the four fish species showed a positive trend with the number of species. The findings can provide a scientific basis for the subsequent application of eDNA technology in the assessment of fish population abundance in natural river systems.