A thin film of hybrid cobalt-manganese hexacyanoferrate (CoMnHCF), a redox mediator was electrodeposited on a glassy carbon (GC) electrode and was employed as an amperometric sensor towards L-Tryptophan (L-Trp). The hybrid film was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction technique (XRD), scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDAX), and electrochemical techniques. The atomic absorption spectroscopic analysis provided the stoichiometry of the hybrid film to be K1.74-y Co-y Mn-0.78 [Fe(CN)(6)], y <= 0.68. The electrochemical impedance study revealed the excellent charge transfer properties of GC/CoMnHCF electrode. The voltammetric investigations demonstrated exceptional electrocatalytic properties of the hybrid film modified electrode when compared to that of bare GC, GC/CoHCF and GC/ MnHCF electrodes, towards the L-Trp oxidation. The kinetic parameters such as electron transfer coefficient, the electron transfer rate constant, the diffusion coefficient and the catalytic rate constant for the electrooxidation process of L-Trp were investigated. The amperometric detection of L-Trp employing GC/CoMnHCF electrode possessed a good sensitivity of 10 x 10(-2) A M-1 cm(-2) in a wide range of detection (2-200 mu M) at a reduced overpotential of 680 mV. In addition, the proposed amperometric method was applied to the detection of L-Trp in commercial milk samples with reproducible results. (C) 2014 Elsevier B.V. All rights reserved.