Neurons release neurotransmitters such as glutamate to communicate with each other and to coordinate brain functioning. As increased glutamate release is indicative of neuronal maturation and activity, a system that can measure glutamate levels over time within the same tissue and/or culture system is highly advantageous for neurodevelopmental investigation. To address such challenges, we develop for the first time a convenient method to realize functionalized borosilicate glass capillaries with nanostructured texture as an electrochemical biosensor to detect glutamate release from cerebral organoids generated from human embryonic stem cells (hESC) that mimic various brain regions. The biosensor shows a clear catalytic activity toward the oxidation of glutamate with a sensitivity of 93 ± 9.5 nA·μM-1 ·cm-2 . It was found that the enzyme-modified microelectrodes can detect glutamate in a wide linear range from 5 μM to 0.5 mM with a limit of detection (LOD) down to 5.6 ± 0.2 μM. Measurements were performed within the organoids at different time points and consistent results were obtained. This data demonstrates the reliability of the biosensor as well as its usefulness in measuring glutamate levels across time within the same culture system.