Abstract A copper microparticle-modified carbon fiber microdisk array electrode was fabricated and employed in capillary electrophoresis for the simultaneous determination of the five aminoglycoside antibiotics (AGs) including netilmicin, tobramycin, lincomycin, kanamycin and amikacin. The array electrode exhibited high catalytic activity for AGs, good reproducibility and stability. Under the optimum separation conditions (separation voltage of 6.2 kV, electrophoretic medium of 125 mM NaOH), the five AGs above were baseline separated within 20 min. At a working electrode potential of 0.7 V (versus saturated calomel electrode), the calibration curves were linear over two orders of magnitude of concentration, and the detection limits ( S/ N=3) were below 2 μM except for lincomycin (6.7 μM). The developed method was successfully employed for the simultaneous determination of the five AGs studied in pharmaceutical injections. The feasibility of this method for the simultaneous determination of lincomycin, kanamycin and amikacin in urine sample was also demonstrated.