Graphene is usually synthesized through deoxygenation of graphene oxide (GO) by hydrazine as the most common and one of the strongest reducing agents. But, due to the high toxicity of hydrazine, it is not a promising reductant in large-scale production of graphene. Here, for the first time, we used melatonin (MLT), as a powerful antioxidant and a biocompatible competitor of hydrazine in reduction of GO suspension. X-ray photoelectron spectroscopy (XPS), current–voltage and optical characteristics of the sheets indicated that the deoxygenation efficiency of the GO suspensions by MLT and under heating in an alkaline condition for 3 h was comparable with the efficiency obtained by hydrazine in similar conditions for 30 min. Based on the XPS analysis, although the amount of nitrogen detected on the surface of the MLT-reduced GO sheets was higher than that of the hydrazine-reduced ones, smaller C–N bonds were formed on the surface of the former. The higher amount of nitrogen on the MLT-reduced GO sheets was assigned to the π–π adsorption of the oxidized MLT molecules on the reduced sheets, which resulted in more stability of the MLT-reduced GO suspension than the aggregation observed in the hydrazine-reduced one, as also confirmed by Raman spectroscopy. A mechanism describing the chemical reduction of the GO sheets by MLT was also proposed. Our results open up a promising outlook for substitution of hydrazine by a safe, biocompatible and powerful reductant for efficient deoxygenation of GO, especially in large-scale production and bio-applications.