The analysis of neurotransmitters in the brain helps to understand brain functions and diagnose Parkinson's disease. Pharmacological inhibition experiments, electrophysiological measurement of action potentials, and mass analysers have been applied for this purpose; however, these techniques do not allow direct neurotransmitter detection with good temporal resolution by using nanometre-sized electrodes. Hence, we developed a method for direct observation of a single neurotransmitter molecule with a gap width of ≤ 1 nm and on the millisecond time scale. It consists of measuring the tunnelling current that flows through a single-molecule by using nanogap electrodes and machine learning analysis. Using this method, we identified dopamine, serotonin, and norepinephrine neurotransmitters with high accuracy at the single-molecule level. The analysis of the mouse striatum and cerebral cortex revealed the order of concentration of the three neurotransmitters. Our method will be developed to investigate the neurotransmitter distribution in the brain with good temporal resolution.