•A novel and sensitive molecularly imprinted kanamycin sensor were developed.•The target analyte detection range and LOD were obtained as 5 nM–1 µM and 5 nM, respectively.•Good precision and accuracy for KAN detection were achieved with no significant interference.•KAN amounts were successfully determined using real milk samples with good recoveries. 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.