Three-dimensional (3D) graphene (GR) microspheres have been successfully prepared for the first time using a simple, easy and green method using β-cyclodextrin aggregates (β-CDAs) as substrates, which could be easily obtained from concentrated aqueous solutions of β-CD. The 3D GR/β-CDAs composites synthesized were characterized using scanning electron microscopy, transmission electron microscopy, ultraviolet/visible spectroscopy, and Raman spectroscopy. A possible formation mechanism was derived. The as-prepared 3D GR/β-CDAs microspheres provided multidimensional electron transport pathways, and this has been exploited in an electrode material for the electrocatalytic oxidation of midecamycin (MD), a widely used macrolide antibiotic. Electrochemical results indicated that the as-prepared 3D GR/β-CDAs microspheres exhibited a higher electrocatalytic activity towards MD oxidation than two-dimensional (2D) GR or β-CDAs, which could be mainly attributed to the improved electrical properties and large surface area of the composite and the high recognition and enrichment capability of β-CDAs. Under optimal conditions, the peak currents on a 3D GR/β-CDA microsphere modified electrode increased linearly with the concentration of MD in the range 0.07–250 μM. The detection limit of MD reached 20 nM (S/N = 3). The present method is promising for the synthesis of high-performance catalysts for sensors, fuel cells and gas-phase catalysis.