Background: Prostate cancer (PCa) ranks second in the incidence of all malignancies in male worldwide. The presence of multiorgan metastases and tumor heterogeneity often leads to unsatisfactory outcomes of conventional radiotherapy treatments. This study aimed to develop a novel folate-targeted nanohydroxyapatite (nHA) coupling to deliver adriamycin (Doxorubicin, DOX), 32 P, and 99m Tc simultaneously for the diagnosis and treatment of prostate-specific membrane antigen (PSMA) positive prostate cancer. Methods: The spherical nHA was prepared by the biomimetic method and characterized. Folic acid (FA) was coupled to nHA with polyethylene glycol (PEG), and the grafting ratio of PEG-nHA and FA-PEG-nHA was determined by the thermogravimetric analysis (TGA) method. In addition, 32 P, 99m Tc, and DOX were loaded on nHA by physisorption. And the labeling rate and stability of radionuclides were measured by a γ-counter. The loading and release of DOX at different pH were determined by the dialysis method. Targeting of FA-PEG-nHA loaded with 99m Tc was verified by in vivo SPECT imaging. In vitro anti-tumor effect of 32 P/DOX-FA-PEG-nHA was assessed with apoptosis assay. The safety of the nano-drugs was verified by histopathological analysis. Results:The SEM images showed that the synthesized nHA was spherical with uniform particle size (average diameter of about 100nm). The grafting ratio is about 10% for PEG and about 20% for FA. The drug loading and the delayed release of DOX at different pH confirmed its long-term therapeutic ability. The labeling of 32 P and 99m Tc was stable and the labeling rate was great. SPECT showed that FA-PEG-nHA showed well in vivo tumor targeting and less damage to normal tissues. Conclusion: FA-targeted nHA loaded with 32 P, 99m Tc, and DOX may be a new diagnostic and therapeutic strategy for targeting PSMA-positive prostate cancer tumors, which may achieve better therapeutic results while circumventing the severe toxic side effects of conventional chemotherapeutic agents.