The use of zinc oxide nanoparticles (ZnO-NPs) across various industries has raised concerns about their potential toxic effects on aquatic organisms, including hybrid grouper. The study aimed to evaluate the toxicological effects of ZnO-NPs exposure on hybrid grouper (±20g) through the analysis of mortality rate, blood performance, organ histopathology, and changes in immune response. The methods involved exposure of ZnO-NPs in four treatment groups (i.e., 0, 50, 100, and 200 ppm) for 96 hours, followed by analyzing the hematology, histopathology, and TNF-α, IL-1β antibody using flow cytometry. The obtained data were analyzed using ANOVA. The results indicated that ZnO-NPs significantly increased fish mortality up to 50% at 200ppm concentration. Significant changes were recorded in hematology, including a 62.96% reduction in hematocrit and a 20.28% increase in leukocyte count. Histopathological analysis showed severe intestinal tissue damage levels reaching 40.43%. Enhanced immune activity expressing TNF-α and IL-1β antibodies, which increased by 15.08% and 6.81%. The highest accumulation of ZnO-NPs was found in the gill organs, with a concentration of 2.94 ppm. This study highlights the toxic risk of ZnO-NPs to hybrid grouper, emphasizing the need for strict monitoring and the development of safety standards to support safer and more sustainable aquaculture practices.