The fabrication of a self-assembled monolayer (SAM) of a cyclopentadienylnickel(II) thiolato Schiff base compound, [Ni(SC6H4NC(H)C6H4OCH2CH2SMe)(η5-C5H5)]2 on a gold electrode is described. Effective electronic communication between the Ni(II) centres and the gold surface was established by electrochemically cycling the Schiff base-doped Au electrode in 0.1 M NaOH from −200 mV to +600 mV. The SAM-modified electrode exhibited quasi-reversible electrochemistry. The integrity of this electrocatalytic SAM, with respect to its ability to block and electro-catalyse certain Faradaic processes, was interrogated using cyclic voltammetric experiments. The formal potential, E°′, varied with pH to give a slope of about −30 mV pH−1. The surface concentration, G, of the nickel redox centres was found to be 1.548×10−11 mol cm−2. By electrostatically doping the SAM using an applied potential of +700 mV versus Ag/AgCl, in the presence of horseradish peroxidase (HRP), it was fine-tuned for amperometric determination of H2O2. The electrocatalytic-type biosensor displayed typical Michaelis–Menten kinetics and the limit of detection was found to be 6.25 mM.