A novel Co₃O₄ nanoparticles-decorated graphene (GR) composite was synthesized by electro-deposition and characterized by scanning electron micrographs, energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy and transmission electron microscopy. Then, amperometric biosensors based on a Co₃O₄ nanoparticles-decorated GR composite modified glassy carbon electrode (GCE) were developed for the sensitive determination of L-tryptophan (Trp). The direct electrooxidation behaviors of Trp on the Co₃O₄/GR/Nafion/GCE were carefully investigated by cyclic voltammetry and square wave voltammetry. The results indicated that Trp showed an increase of the oxidation peak current with a negative shift of the oxidation peak potential compared with that on the bare GCE. Under optimum conditions, the proposed biosensor can be applied to the quantification analysis of Trp with a wide linear range covering 0.05–10 μM (R = 0.996) and a low detection limit of 0.01 μM. The experimental results also showed that the sensor exhibited good reproducibility, long-term stability as well as high selectivity. Moreover, the novel biosensor for the detection of Trp in a real amino acid sample with satisfactory results has been proved.