A novel and sensitive voltammetric method was proposed for separation and determination of glutathione (GSH) and L‐tyrosine (Tyr) at acetylene black and chitosan modified glassy carbon electrode (AB‐CS/GCE). By introducing chlorogenic acid (CGA) as a new electrocatalytic mediator, GSH could be detected at much lower potential with symmetric peak shape. Acetylene black and chitosan composite served as current signal amplifier for sensitive detection. The electrochemical behavior of GSH and Tyr in the presence of CGA was studied at AB‐CS/GCE and complete separation of anodic peaks was achieved. Under the optimum conditions, the electrocatalytic oxidation peak current of GSH showed a linear dependence on its concentration in the ranges of 2.0×10−7‐4.0×10−5 M with the detection limit of 5.8×10−8 M (S/N=3), while the oxidation peak current of Tyr was linear to its concentration from 2.5×10−6 to 4.3×10−4 M with the detection limit of 9.2×10−7 M (S/N=3) by differential pulse voltammetry (DPV). The established method has been applied to the simultaneous determination of GSH and Tyr in human urine with satisfactory results.