Brain-on-Chip devices, which enable on-chip cultures of neurons to simulate brain functions, are receiving tremendous attention from both fundamental and clinical research. Thus, microsensors are being developed to accomplish real-time monitoring of neurotransmitters, which are the benchmarks for neuron network operation. Electrochemical sensors are promising candidates for detecting a critical neurotransmitter, dopamine. However, current state-of-the-art electrochemical dopamine sensors are suffering from issues like limited sensitivity and cumbersome fabrication. Here, we report a novel route in monolithically microfabricating vertically aligned carbon nanofiber electrochemical dopamine microsensors with an anti-blistering slow cooling process. Thanks to the microfabrication process, we can achieve microsensors with complete insulation and large surface areas. The champion device shows extremely high sensitivity of 4.52×104 µA/µM·cm2 , which is two-orders-of-magnitude higher than current devices, and a highly competitive limit of detection of 0.243 nM. These figures-of-merit will open new windows for applications such as electrochemical recording from a single neuron.