The increasing pollution from non-biodegradable antibiotics poses a significant threat to ecosystems and human health. TiO2 is widely acknowledged as the premier photocatalyst for removing antibiotics from wastewater. To address the limitations of TiO2 in wide forbidden band width and low efficiency separation/migration of carriers, N-doping and oxygen vacancies (OVs) are co-modified within TiO2 nanosheets through the one-step calcination of a mixture of H2TiO3 and urea. The results indicate that the co-modified sample (TiO2-U5) exhibited the highest TC removal efficiency of 90% after 60 minutes of reaction, with a kinetic constant 3.6 times higher than that of pristine TiO2. The effective mineralization of TC is confirmed by three-dimensional excitation-emission matrix fluorescence spectroscopy (3D EEMs). Systematic characterization of the photocatalyst structure and monitoring of intermediates/products reveal that the synergistic effect of N-doping and oxygen vacancies (OVs) enhances band alignment, light response, and carrier separation/migration, thus improving the photocatalytic degradation performance of TC. This study highlights the significant potential of adjusting charge transport and enriching active sites through the co-introduction of doping and surface defects on TiO2 for the photocatalytic degradation of antibiotics.