Conserving aquatic ecosystems requires efficient tools to accurately assess the biodiversity of aquatic species. However, existing knowledge is insufficient in terms of the reliability and the comparability of methods measuring fish diversity. Environmental DNA (eDNA), as a promising method, was used to detect fish taxa in this study. We optimized the eDNA method in the laboratory, and applied the optimal eDNA method to survey fish diversity in a natural aquatic life reserve. We simulated necessary steps of the eDNA method in the lab to increase the confidence of the field survey. Specifically, we compared different eDNA sampling, extraction, and sequencing strategies for accurately capturing fish species of the target area. We found that 1L water samples were sufficient for sampling eDNA information of the majority taxa. The filtration was more effective than the centrifugal precipitation for the eDNA extraction. The cloning sequencing was better than the high-throughput sequencing. The field survey showed that the Shannon–Wiener diversity index of fish taxa was the highest in Huairou Reservoir. The diversity index also showed seasonal changes. The accuracy rate of detecting fish taxa was positively correlated with the eDNA concentration. This study provides a scientific reference for an application of the eDNA method in terms of surveying and estimating the biodiversity of aquatic species.