Brain organoids and assembloids have been developed to investigate brain-associated diseases, such as neurodegenerative diseases and brain cancer. However, brain assembloid-based in vitro drug evaluation models remain limited due to the lack of three-dimensional (3D) electrophysiological sensors that are capable of continuously monitoring the changes in drug-induced electrophysiological signals. In this study, we developed a 3D electrophysiological sensor by integrating glioblastoma multiforme (GBM)-midbrain (MB) assembloids with dual-channel flexible gold (Au) nanodot-patterned electrodes, for in vitro drug evaluation with continuous monitoring. Au nanodot-patterned films were fabricated using laser interference lithography and attached to a polymer-based 3D concave mold to form flexible Au nanodot-patterned electrodes, which enhanced electrophysiological signal detection. The GBM-MB assembloid was generated by integrating GBM and MB organoids within a neurotrophin factor-3 (NT3)/gold nanoparticle (GNP)-incorporated hydrogel, which improved electrical conductivity and neural connectivity within the assembloid, leading to increased spike activity. The 3D electrophysiological sensor, consisting of the assembloid on dual-channel flexible electrodes, enabled continuous and independent detection of electrophysiological signals from individual organoids. Both organoids showed increased cancer-induced electrophysiological signals, which were significantly reduced by treatment with Everolimus (a cancer drug), validating the system’s effectiveness for dynamic drug evaluation. Electrophysiological signal analysis further revealed the influence of GBM organoid states on MB organoids. The proposed electrophysiological sensors, integrating continuous monitoring of brain assembloid activity with dual-channel flexible electrodes, offers a promising platform for toxicity assessment and drug screening for diverse brain diseases.