In this work, molecularly imprinted kanamycin (KAN) electrodes were prepared using electrochemical polymerization of pyrrole (Py). First, a glassy carbon electrode was coated with an optimized volume of graphene oxide (GC/GO) to provide a high surface area electrode. Py is then polymerized on GC/GO electrode using cyclic voltammetry in the presence of KAN following by KAN removal using HCl (GC/GO-pPy-KAN*). Electrode preparation steps were also optimized using microscopic, spectroscopic, and electrochemical methods. Finally, the analytical performance of the prepared GC/GO-pPy-KAN* electrode was investigated for the determination of KAN. The limit of detection and the detection range was calculated as 5 nM and 5 nM–1 µM, respectively. The precision, accuracy, and interference studies showed good precision and relative error with minimum interference for the chosen substances. Moreover, real sample analysis was also performed using 4 different milk samples with good recovery values. Consequently, a novel, simple, and sensitive sensor was developed using an easy and low-cost fabrication method for the detection of KAN in food samples such as milk.