The invention discloses a method for monitoring and evaluating fish passage effects based on environmental DNA. The method comprises the following steps: S1, setting three sampling points at an entrance, exit and rest pool of a fish passage, and collecting water samples and performing storage; S2, performing suction filtration on the collected water samples, and then performing extraction of totalenvironmental DNA; S3, using the extracted total environmental DNA as a template, and using designed primers to PCR amplify a 16S rDNA sequence to obtain a PCR product; S4, recovering and purifying the PCR product, and performing high-throughput sequencing to obtain a PCR product sequence; S5, establishing a fish 16S rDNA sequence alignment database; S6, searching for homologous sequences in theNCBI database and the established database through BLAST, and determining fish species information; and S7, counting the obtained fish species information, and completing the monitoring and evaluationof the fish passage effects. Compared with other existing methods, the method does not need to carry out a large number of fishing operations or instrument operations, is simple in sampling, cannot cause harms to fish, and thereby is conducive to the protection of fish.