Cu Se nanoparticles (Cu Se NPs) are widely used for optical diagnostic imaging and photothermal therapy due to their strong near-infrared (NIR) optical absorption. With the continuous expansion of applications using Cu Se NPs, their biosafety has received increasing attention in recent years. Cu Se NPs can enter the brain by crossing the blood-brain barrier, but the neurotoxicity of NPs remains unclear. The present investigation provides direct evidence that the toxicity of Cu Se NPs can be specifically exploited to kill rat pheochromocytoma PC-12 cells (a cell line used as an model for brain neuron research) in dose- and time-dependent manners. These cytotoxicity events were accompanied by mitochondrial damage, adenosine triphosphate (ATP) depletion, production of oxidizing species (including reactive oxygen species (ROS), malondialdehyde (MDA) and hydrogen peroxide (HO)), as well as reductions in antioxidant defense systems (glutathione (GSH) and superoxide dismutase (SOD)). Moreover, our study also confirmed that Cu Se NPs markedly induced neurotoxicity and oxidative stress damage in the striatum and hippocampal tissues of BALB/c mice. These findings suggest that Cu Se NPs induce neurotoxicity in PC-12 cells and BALB/c mice oxidative stress damage, which provides useful information for understanding the neurotoxicity of Cu Se NPs.