Environmental pollutant nanoplastics (NPs) cross the blood–brain barrier and induce central nervous system toxicity through multiple pathways, with oxidative stress as the core neurotoxicity initiator. However, the brain penetration depth of NPs and associated oxidative stress distribution have not yet been investigated. Herein, we developed a flexible electrochemical sensor based on platinum nanoparticle-modified carbon nanotube fiber (Pt/CNF) to minimize insertion damage. The sensor exhibits excellent mechanical compliance and high sensitivity for hydrogen peroxide (H₂O₂) detection. Using this sensor for real-time in situ H₂O₂ monitoring in human cortical organoids (COs), we systematically investigated the oxidative stress levels at depths of 100 and 300 µm in COs exposed to polystyrene nanoplastics (PS-NPs) for different durations within 6 days. Our results demonstrate that oxidative stress at the same depth increased with longer exposure time, and showed distinct spatial locality, concentrating primarily in the ~ 100 µm-deep penetration region. This study quantifies the spatiotemporal neurotoxicity of nanoplastics in human brain models and provides a robust technical framework for environmental health risk assessment in other tissues.