Copper is an important heavy metal and an essential trace element for aquatic organisms and is found at low concentrations in aquatic ecosystems. In aquaculture, copper sulfate is used as a therapeutic agent to reduce parasitic infections in cultured fish. However, exposure to heavy metals beyond a safe concentration range can cause a series of physiological and biochemical stresses in aquatic animals, including death. Fish are generally most sensitive to the effects of Cu2+ exposure during early life stages, especially during embryonic and larval development, which can result in reduced fertilization success, chromosomal abnormalities, DNA injury, increased incidence of yolk membrane ruptures, reduced hatching success and survival, altered time to hatching, embryonic and larval teratogenicity, inhibited growth, abnormal larval behaviors. Yellowtail kingfish (Seriola aureovittata) is a fast-growing marine species widely distributed in temperate and subtropical regions of the oceans. It has a large body size, superior flesh quality, high economic value, and can be cultured in sea cages, indoor recirculating aquaculture systems, aquaculture crafts, and other culture modes. Therefore, S. aureovittata is a promising candidate for open-ocean aquaculture in China. To investigate the effects of copper exposure on the early life stages of S. aureovittata, embryos and yolk sac larvae were exposed to copper concentrations of 0, 0.034, 0.060, 0.107, 0.192, 0.340, 0.600, and 1.070 mg/L for 60 h and 5 d under laboratory conditions in the present study. The half-lethal concentration (LC₅₀) and safe concentration (SC) were determined for embryos and newly hatched larvae of S. aureovittata, respectively. In addition, differential gene expression patterns and related signaling pathways were investigated in embryos and yolk-sac larvae to elucidate possible molecular mechanisms. The results showed that: (1) Low hatching rate of embryos, high malformation rate of newly hatched larvae, and high utilization speed of yolk sacs during the early developmental stage of S. aureovittata under copper exposure stress. (2) The 48 h-LC50 of copper for S. aureovittata embryos was 0.08 mg/L, which was much lower than that of yolk sac larvae (48 h-LC50 = 0.60 mg/L). The SC of Cu²⁺ in S. aureovittata yolk sac larvae was 0.034 mg/L. (3) Transcriptome sequencing analysis revealed that differentially expressed genes (DEGs) were mainly enriched in the oxidative phosphorylation pathway during the embryonic development stage of S. aureovittata, whereas in the yolk sac larval stage, DEGs were mainly enriched in the cell cycle and phototransduction pathways, indicating that excessive copper exposure may injure the antioxidative ability of embryos and cause DNA injury and phototransduction inhibition in larvae. The present study determined the safe concentration of copper sulfate during the early developmental stages of S. aureovittata and revealed the molecular responses of embryos and yolk sac larvae to copper exposure stress. These results could provide theoretical support for the scientific and staged application of copper agents in the aquaculture of S. aureovittata and technical guidance for monitoring aquaculture and marine environments.