CuO NPs are nanomaterials with catalytic activity and unique thermo-physical properties used in different fields such as sensors, catalysts, surfactants, batteries, antimicrobials and solar energy transformations. Because of its wide field of use, these nanoparticles accumulate in the aquatic environment and thus lead to toxic effects on aquatic organisms. The toxicological findings about CuO NPs are controversial and these effects of CuO NPs on aquatic organisms have not been elucidated in detail. Therefore, the aim of this study was to investigate the toxic effect of CuO NPs on zebrafish embryos using different parameters including molecular and morphologic. For this purpose, zebrafish embryos at 4 h after post fertilization (hpf) were exposed to different concentrations of CuO NPs (0.5, 1, 1.5 mg/L) until 96 hpf. Mortality, hatching, heartbeat, malformation rates were examined during the exposure period. In addition, Raman spectroscopy was used to determine whether CuO NPs entered into the tissues of zebrafish larvae or not. Moreover, the alterations in the expression of genes related to the antioxidant system and innate immune system were examined in the embryos exposed to CuO NPs during 96 h. The results showed that CuO NPs was not able to enter into the zebrafish embryos/larvae tissues but caused an increased the mortality rate, a delayed hatching, and a decreased heartbeat rate. Moreover, CuO NPs caused several types of abnormalities such as head and tail malformations, vertebral deformities, yolk sac edema, and pericardial edema. RT-PCR results showed that the transcription of mtf-1, hsp70, nfkb and il-1β, tlr-4, tlr-22, trf, cebp was changed by the application of CuO NPs. In conclusion, short-term exposure to CuO NPs has toxic effects on the development of zebrafish embryos.