Ultrafine CuO nanoparticles (UCNs) were well-isolated in the nano-channels of ordered mesoporous carbon (OMC) via a double solvent procedure. In this approach, uniform OMC meso-channels of large surface area acted as nanoreactors embedded with small nanoparticles. n-Hexane was used as hydrophobic solvent to disperse OMC, and hydrophilic Cu(NO₃)₂ aqueous solution served as a precursor to fill the nano-channels. The interfacial force between double solvents of Cu(NO₃)₂ and n-hexane facilitated the precursor filling within the OMC mesopores, and the subsequent heat treatment triggered in situ isolation of UCNs within OMC porous channels. This final product was denoted as UCNs@OMC, and the structure and morphology of UCNs@OMC were characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and N₂-sorption. Increased glucose oxidation currents implied the accessibility of the active catalytic sites of UCNs@OMC, and an UCNs@OMC based glucose sensor was fabricated and tested exhibiting good reproducibility and selectivity.