Abstract: Environmental DNA (eDNA) metabarcoding is a high-efficiency, high-sensitivity and non-invasive species investigation tool. At present, there are many studies on the investigation of fish diversity based on eDNA metabarcoding which however is not well-developed, with a lack of consensus on the actual use of different primers. In order to reduce the cost of sequencing and screen out the universal primers with the best practical effect, we selected the water samples from eight sites in Sanya fish markets and Atlantis Aquarium, then compared the differences of three sets of universal primers (MiFish-U, AcMDB07 and Ac12S) for fish eDNA. The results show that: 1) There were significant differences in the reads number after quality control, the fish reads number, the total Operational taxonomic units (OTUs) number, the fish OTUs number and the ratio of fish reads among three sets of primers. MiFish-U had the highest amplification efficiency and targeting to fish species; 2) MiFish-U had the highest number of species (140 species), while AcMDB07 and Ac12S had 128 and 97 species, respectively; 3) The reference databases of Ac12S and AcMDB07 were not perfect, and 72.76% and 42.11% of the OTUs belonging to Ac12S and AcMDB07 could not be annotated to the species level, respectively; 4) There are very few endemic fish detected by Ac12S (Only four species), suggesting that it was easier to be replaced by the other two sets of primers in the actual use process, and MiFish-U had the lowest substitutability; 5) The three sets of primers reflected similar general trends in the fish abundance, but there were some differences in the specific species. The results show that MiFish-U is superior to AcMDB07 and Ac12S in species detection, considering various factors such as OTUs annotation, especially the conditions of existing reference data.