An asymmetric diimine Schiff base, 2-((4-((2-hydroxybenzylidene) amino) benzylidene) amino) phenol (2-HBP), was covered to a glassy carbon (GC) electrode surface in nonaqueous medium to fabricate a modified electrode. Cyclic voltammetry was applied for the surface modification experiments in 1 mM diimine solution. Cyclic voltammetry, contact angle measurement, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques were employed to characterize the modified GC (2-HBP-GC) surface. Electrochemical impedance results showed that the modified surface blocked the electron transfer with a 98.9% surface coverage value. Additionally, theoretical study was performed to gain insight into the structural and tautomeric aspects of the asymmetric diimine Schiff base. The phenol-imine and ketoamine tautomerism forms of the asymmetric diimine Schiff base were investigated by using DFT calculations.