The detection of nucleobases is critical for enhancing DNA sequencing technologies. This study employs density functional theory (DFT) and nonequilibrium Green’s function (NEGF) methods to explore the adsorption behavior of natural DNA bases (adenine (A), thymine (T), guanine (G), and cytosine (C)) on a C2O monolayer and to assess its electronic transport properties. Our results show that adsorption occurs predominantly via physisorption, characterized by minimal charge transfer and weak dispersion interactions, leading to the emergence of flat molecular bands near the conduction and valence band edges. Current–voltage (I–V) measurements reveal that current onset occurs around 2.5 V, with guanine exhibiting the highest current and cytosine causing the largest current reduction compared to the pure C2O monolayer. Sensitivity analysis indicates that at 3.0 V, adenine achieves the highest current sensitivity (∼48%), while at 3.5 V, cytosine reaches peak sensitivity (∼60%). These sensitivity trends enable selective differentiation of nucleobases by tuning the applied voltage, highlighting the potential of C2O monolayer-based nanodevices for voltage-dependent and -selective DNA base detection.