Silver carp (Hypophthalmichthys molitrix) is an important freshwater economic fish in China’s aquaculture industry. In 2020, six species of freshwater farmed fish in China had production greater than a million tons, of which the yield of silver carp was 3.8129 million tons, ranking second. Silver carp is widely distributed in natural waters, and plays a major role in regulating the ecological environment. In the process of H. molitrix culture, hypoxia has become an important factor restricting the increase of its yield. In previous studies, miR-17a-5p was identified and differentially expressed in H. molitrix under hypoxia stress based on the small RNA sequencing. The micro-ribonucleic acids (miRNAs), a class of endogenous and noncoding small RNA, play an important role in biological resistance to environmental stresses. In order to explore the role of miR-17a-5p in H. molitrix under hypoxia, sequence analysis and target gene prediction of miR-17a-5p was performed. The results showed that the miR17a-5p are highly conserved among species, and 381 target genes were identified, including HIF-1α. Dual-luciferase reporter assay results demonstrated that when 293T cells were co-transfected with miR17a-5p mimics and WT-pmirGLO-HIF-1α 3′UTR, the luciferase activity decreased, confirming HIF-1α to be a target gene of miR17a-5p. This conclusion is in agreement with the observation of the increasing trend of HIF-1α alongside the decreasing pattern of miR17a-5p under hypoxic stress in different tissue. KEGG pathway analysis revealed that the target genes were significantly enriched in Sulfur metabolism, mTOR signaling pathway and Terpenoid backbone biosynthesis. The expression of 11 target genes involved in the above three signaling pathways under hypoxia in liver, brain, heart and gill were detected by quantitative real-time PCR. The results showed that the overall trend of increase for the relative expressions of these genes, and upregulation was most pronounced in liver. In this study, we analyzed the bioinformatics of miR-17a-5p, predicted and further identified its target genes, and subjected H. molitrix to different degree of hypoxia stress, revealing that the downregulation of miR-17a-5p expression in H. molitrix under hypoxia stress attenuates its inhibitory effect on its target genes. Our finding provides a new idea for miR-17a-5p regulation of its target genes in H. molitrix under hypoxia stress, and provides a reference for further analysis of the role of miRNAs in the process of hypoxia tolerance of fish.