Here, we design and synthesize a novel 2D Cu-tetrakis(4-carboxyphenyl)porphyrin (TCPP) metal-organic framework (MOF) sheet and ultrasmall CuO nanoparticle (CuO USNP) hybrid (Cu-TCPP MOF/CuO nanocomposite). The graphene-like ultrathin Cu-TCPP MOF sheets offer high surface-to-volume atom ratios and many active sites, which is beneficial for loading more CuO USNPs. The CuO USNPs with ultrasmall size (<5 nm) have promising conductivity and excellent enzymatic ability for HO. The successfully prepared nanocomposites are characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR) techniques. The 2D graphene-like ultrathin Cu-TCPP MOF sheets show no HO-sensing signals, whereas CuO USNPs exhibit a clear reduction peak for detection of HO. Interestingly, the combination of two kinds of nanomaterials improved the HO sensing ability due to their synergistic effect. The properties of the unmodified electrodes and the Cu-TCPP MOF/CuO nanocomposite-modified electrodes were systemically studied by cyclic voltammetry (CV), current-time (i-t) response, and square-wave voltammetry (SWV) techniques. The electrochemical sensor for the detection of HO based on the Cu-TCPP MOF/CuO nanocomposite has a lower detection limit of 0.13 μmol·L and wider linear range of 0.1 × 10 ~ 0.59 × 10 mol·L and 1.59 × 10 ~ 20.59 × 10 mol·L when compared with the CuO USNPs-modified electrode. The electrochemical sensor can be further used to detect HO produced by cells. Graphical abstract The mechanism for sensing HO produced from cells based on a Cu-TCPP MOF/CuO USNPs nanocomposite-modified electrode.