A glassy carbon electrode modified with a composite consisting of electrodeposited chitosan and carboxylated multi-walled carbon nanotubes (e-CS/MWCNTs/GCE) was used as a working electrode for simultaneous determination of dopamine (DA), serotonin (5-HT) and melatonin (MT), which were related to circadian rhythms. The electrochemical characterizations of the working electrode were carried out via electrochemical impedance spectroscopy and chronocoulometry. It was found that electrochemical modification method, that was cyclic voltammetry, may can cause continuous CS polymerization on MWCNTs surface to form a dense membrane with more active sites on the electrode, and the electrochemically active surface area of e-CS/MWCNTs/GCE obtained was about 7 times that of GCE. The electrochemical behaviour of DA, 5-HT and MT on working electrode were carried out via differential pulse voltammetry and cyclic voltammetry. The results showed that e-CS/MWCNTs/GCE solved the problem that the bare electrode could not detect three substances simultaneously, and can catalyze oxidation potential difference as low as 0.17 V of two substances reaction at the same time, indicating very good electrocatalytic activity. By optimizing the detection conditions, the sensor showed a good linear response to DA, 5-HT and MT in the range of 20-1000 μmol/L, 9-1000 μmol/L and 20-1000 μmol/L, and the detection limits were 12 μmol/L, 10 μmol/L and 22 μmol/L (S/N = 3), respectively. In addition, the proposed sensor was successfully applied to the simultaneous detection of DA, 5-HT and MT in human saliva samples.