This paper describes the role of various external redox mediators in biodetector systems using bioinspired nanomaterials based on magnetite@polydopamine (Fe₃O₄@PDA), and their influence on the working parameters of the designed systems. The investigated mediators were hydroxy(methylferrocene, 1,4-benzoquinone, methylene blue, hydroquinone, and [Fe(CN)₆]^3-/4- redox couple. The amperometric glucose biosensor was fabricated by immobilizing glucose oxidase from Aspergillus niger (GOx) onto the Fe₃O₄@PDA nanomaterial, with the addition of a strictly defined amount of a specific external mediator. Among all tested mediators, the most favorable results were obtained for hydroxymethyl(ferrocene) with the integrated GC/Fe₃O₄@PDA-GOx system. The mediator exhibited a high linear amperometric response in a glucose concentration range from 1 to 20 mM, with a relatively high sensitivity of 139.71 μAmM⁻¹ cm⁻² and a limit of detection of 1.54 μM. Moreover, the system showed good stability over time, reproducibility, and resistance to interference.