Near-infrared (NIR) long lasting persistent luminescence nanoparticles (PLNPs) have attracted considerable attention in the area of in vivo bioimaging, due to their background-free luminescence characteristics and deep tissue penetration. However, the low fluorescence quantum yield and short afterglow of the currently available PLNPs limit their applications. Here, water-soluble Cr3+-doped ZnGa2O4 PLNPs with the highest quantum yield (η = 20%) ever reported, bright NIR emission, and excellent colloidal stability were successfully prepared by a one-step hydrothermal method. The afterglow of the resultant nanocrystals lasted for more than 5 days and could be repeatedly reactivated by the light (λ = 657 nm) of a portable light emitting diode lamp after decay. These nanocrystals were functionalized with α,ω-dicarboxyl-terminated poly(ethylene glycol) and poly(acrylic acid) to improve their stability and biocompatibility, so that they could be conjugated with a c(RGDyK) peptide and labeled with 99mTc for targeted imaging of orthotopic breast cancer by afterglow luminescence imaging and single-photon emission computed tomography imaging. Our NIR-PLNP probes can effectively avoid tissue auto-fluorescence and the light scattering caused by continuous excitation during the diagnosis of cancer.