A rapid and simple procedure was developed for the preparation of a highly stable and leach-proof glucose oxidase (GOx)-bound glassy carbon electrode (GCE). Crosslinked GOx via glutaraldehyde was drop-cast on a KOH-pretreated GCE followed by drop-casting of 3-aminopropyltriethoxysilane (APTES) to form a stable bioactive layer. At -0.45 V, the biosensor exhibited a wide dynamic detection range of 0.5-48 mM for commercial glucose and 1.3-28.2 mM for Sugar-Chex blood glucose linearity standards. Several endogenous electroactive substances and drug metabolites commonly found in blood were tested and provoked no signal response. To our knowledge, the developed procedure is the most rapid method for preparing a glucose biosensor. The biosensor suffered no biofouling after 7 days of immersion in Sugar-Chex blood glucose. With excellent production reproducibility, GOx-bound electrodes stored dry at room temperature retained their initial activity after several weeks.