In this paper, we report the results of the surface oxidation of platinum (Pt) microwires in aqueous sulfuric acid (H₂SO₄) solutions by using a cyclic voltammetry technique. The Pt microwire chips were scanned and applied with voltage potentials ranging from 0 to 1.4 V in the H₂SO₄ solution with concentrations from 0.0003 to 0.0018 M to find out the optimized concentration of sulfuric acid for the oxidation process. The cyclic voltammetry (CV) measurements show the oxidation peak at a potential range from 1.1 to 1.2 V. This is the peak of the interfacial place exchange of chemisorbed O (O_chem) and surface Pt atoms, resulting in the formation of a quasi-3D surface PtO lattice comprising Pt²⁺ and O²⁻. The oxidized surface Pt microwires were then functionalized with a 3-aminopropyl triethoxy silane (APTES) and glucose oxidase (GOD) was immobilized onto the functionalized chips for further application in glucose detection. By using this process, Pt microwires have been used for the successful detection of glucose in solution with concentrations in the range of 4–20 mM.