A simple, green and sensitive method is described for voltammetric determination of midecamycin (MD), a widely used macrolide antibiotic, based on its electrochemical oxidation at a graphene-based electrochemical sensing platform by means of electrochemical reduction of graphene oxide (GO) on a glassy carbon electrode (GCE). Compared with bare GCE, the graphene modified GCE exhibited excellent enhancement effect on the electrochemical oxidation of MD. A well-defined oxidation peak of MD occurred at 0.69 V in 0.1 mol L⁻¹ phosphate buffer solution (PBS) of pH 7.4. Under optimized conditions, the anodic peak current was linear to the concentration of MD in the range from 3.0 × 10⁻⁷ to 2.0 × 10⁻⁴ mol L⁻¹ with the detection limit of 1.0 × 10⁻⁷ mol L⁻¹. The relative standard deviation (RSD) was 2.32% for 8.0 × 10⁻⁶ mol L⁻¹ MD (n = 10). To further validate its possible application, the proposed method was successfully used for the determination of MD in pharmaceutical formulations and biological fluids with satisfactory results.