Locating nanotherapeutics at the active sites, especially in the subcellular scale, is of great importance for nanoparticle-based photodynamic therapy (PDT) and other nanotherapies. However, subcellular targeting agents are generally nonspecific, despite the fact that the accumulation of a nanoformulation at active organelles leads to better therapeutic efficacy. We herein designed a PDT nanoformulation by using graphene oxide quantum dots (GOQDs) with rich functional groups as both the supporter for dual targeting modification and the photosensitizer for generating reactive oxygen species, and upconversion nanoparticles (UCNs) as the transducer of excitation light. A tumor-targeting agent, folic acid, and a mitochondrion-targeting moiety, carboxybutyl triphenylphosphonium, were simultaneously attached onto the UCNs-GOQDs hybrid nanoparticles by surface modification, and a synergistic targeting effect was obtained for these nanoparticles according to both in vitro and in vivo experiments. More significant cell death and a higher extent of mitochondrion damage were observed compared to the results of UCNs-GOQDs nanoparticles with no or just one targeting moiety. Furthermore, the PDT efficacy on tumor-bearing mice was also effectively improved. Overall, the current work presented a synergistic strategy to enhance subcellular targeting and the PDT efficacy for cancer therapy, which may also shed light on other kinds of nanotherapies.