Next-generation photodynamic therapy (PDT) is envisaged to be based on light-activated photosensitizers with small sizes and high performance, eradicating microbial and deadly cancer cells without harming healthy cells. Here, nitrogen-doped graphene quantum dots (N-GQDs) are hydrothermally synthesized and composited with TiO₂ nanoparticles (NPs). The resulting N-GQDs/TiO₂ nanocomposite is then examined as a PDT photosensitizer with an average size of 21 nm. Reactive oxygen species (ROS) production by photo-excited N-GQDs, TiO₂, NPs and N-GQDs/TiO₂ nanocomposite is investigated using anthracene and methylene blue as chemical probes for the identification of singlet oxygen and hydroxyl radical. The ROS-production ability of the nanocomposite is found to be considerably higher than that of N-GQDs and TiO₂ NPs, achieving reduction rates of 94% and 93% in the absorption intensity of anthracene and methylene blue under UVA irradiation for 75 and 60 min, respectively. The higher ROS production is attributed to the efficient energy transfer from N-GQDs to TiO₂ NPs due to the fluorescence resonance energy transfer effect as well as a reduction in the recombination of photogenerated electron–hole pairs. Furthermore, afterglow emission intensity of the nanocomposite irradiated by UVA light slightly changes after 360 s. Alternatively, no decrease is observed in the absorption intensity of the chemical probes in the presence of N-GQDs/TiO₂ nanocomposite under no irradiation, indicating its lack of dark toxicity. Therefore, the proposed biocompatible TiO₂-based nanocomposite with long-lived afterglow, intense photoluminescence, and high ROS production ability can be employed as a photosensitizer for cancer treatment using PDT.