A homoleptic ionic Cu(I) coordination complex that was based on 2,2′-biquinoline ligand functionalized with long alkyl chains (Cu(I)–C18) was used as a precursor to modify a carbon nanofiber paste electrode (Cu–C18/CNF). Randomized copper oxide microelectrode arrays dispersed within carbon nanofiber paste (CuOx/CNF) were obtained by electrochemical treatment of Cu–C18/CNF while using cyclic voltammetry (CV). The CuOx/CNF exhibited high electrocatalytic activity towards glucose oxidation at +0.6 V and +1.2 V vs. Ag/AgCl. Infrared Spectroscopy (FTIR) and scanning electron microscopy (SEM) characterized the electrodes composition. Cyclic voltammetry (CV), square wave-voltammetry (SWV), and multiple-pulsed amperometry (MPA) techniques provided optimized conditions for glucose oxidation and detection. A preconcentration step that involved 10 minutes accumulation at open circuit potential before SWV running led to the lowest limit of detection and the highest sensitivity for glucose detection (5419.77 µA·mM−1·cm−2 at + 1.1 V vs. Ag/AgCl) vs. Cu-based electrodes reported to date in literature.