Metal-oxide nanoparticles (NPs), including copper oxide (CuO-NPs), are being released uncontrollably into the environment, posing a significant threat to aquatic life. Chlorella vulgaris (ChV) demonstrated a protective effect against hazardous substances by considerably minimizing oxidative burden caused by many various compounds including NPs. This study planned to assess the effect of sub-acute exposure to CuO-NPs on neurobehavioral impairments in Oreochromis niloticus (O. niloticus) fish, and assess the ameliorative role of ChV against the induced neurotoxicity. A total of 144 O. niloticus was equally grouped into four groups as follows: control (C), ChV (30gm/kg diet), CuO-NPs (1/10 LC50, 5.72 µg/L), and CuO-NPs/ ChV groups. Our data proved that the exposure to CuO-NPs induced a significant increase of hyperpigmentations, ulcerations, tail corrosions and hemorrhages. A significant increase of surfacing, hiding, loss of equilibrium, laterality and motionless behaviors was recorded in CuO-NPs exposed fish. From the contrary side, exposure to CuO-NPs significantly decreased the number of crossings, feeding behaviors, escape and knocking reflexes. The CuO-NPs exposed fish revealed a significant reduction in total antioxidant capacity (TAC) level, and superoxide dismutase (SOD) activity, while malondialdehyde (MDA) and 8-hydroxyguanosine (8- OH2dG) levels significantly increased in brain tissue, in addition to a decline in acetylcholine esterase (AchE) activity in serum. The RT-PCR analysis demonstrated an up-regulation of nuclear factor kappa B (NF- 𝜅β) and Caspase-3 mRNA expression levels, while b-cell lymphoma 2 (Bcl-2) mRNA expression level down-regulated in brain tissue. Moreover, histopathological alterations were observed in the brain tissue. Importantly, ChV significantly protected fish from neurobehavioral impairments induced by CuO-NPs. Our data demonstrated the neuroprotective effects of ChV in CuO-NPs exposed fish, promoting ChV's usage as a potential anti-inflammatory, anti-oxidant and neuroprotective agent.