This study aimed to investigate the transformation of copper oxide nanoparticles (CuO NPs) in aquatic environments under different ionic strength and further examine its effects on copper toxicity and bioaccumulation by monitoring the responses and uptake behaviours of zebrafish embryo. Ionic strength (IS) was simulated according to surface water (1.5 mM), groundwater (15 mM), and wastewater (54 mM), representing low-, mid-, and high-IS water, respectively. At the highest exposure of 10 mg CuO/L, zebrafish larvae mortality was increased from 21.3% to 33.3%, when IS decreased from 54 to 1.5 mM. Low-IS solution also caused the highest numbers of delayed hatching embryo (81.3%) and opaque yolk deformation (36.3%). Copper bioaccumulation markedly increased when larvae were exposed to low-IS water (35%) relative to high-IS water (15%). Exposing to low-IS particularly enhanced copper uptake (~15 ng Cu/g inside embryo), facilitating the copper accumulation in the heart of larvae, whereas aggregated CuO NPs (>500 nm) in mid- and high-IS water were blocked from the embryo and found abundantly in the body axis and tail. Results indicate that CuO NPs in low-IS solutions rapidly form the relatively small CuO NP aggregates with a high copper dissolution, which would pose great concern for aquatic organisms.