〔Abstract〕 Objective To explore a non-invasive assay for screening osteosarcoma patients with positive programmed death receptor-1(PD-1)/programmed death receptor ligand-1(PD-L1) signaling pathway expression. Methods The subcutaneous tumor bearing mouse model of human Osteosarcoma cells(OS-732) was established by the method of tumor formation, and toxicity test was performed to verify the toxicity of PD-L1 antibody to mouse organs.124I-anti-PD-L1 monoclonal antibody molecular probe was further synthesized, and OS-732 rats were injected with 18.5 MBq124I-anti-PD-L1 probe in tail vein to start OS-732 osteosarcoma Micro Positron Emission Tomography/Computed Tomography(Micro-PET/CT) imaging. Results OS-732 osteosarcoma model was successfully constructed, immunohistochemistry confirmed the presence of PD-L1 expression in OS-732 osteosarcoma, and Micro-PET/CT imaging was successfully performed at different time points(2, 24 and 48 h after probe injection) to achieve non-invasive real-time observation of PD-L1 in OS-732 osteosarcoma. Conclusion In this study, the124I-anti-PD-L1 monoclonal antibody molecular probe was constructedin vitro, and Micro PET/CT imaging verified that the probe successfully targeted the PD-L1 receptor of OS-732 Osteosarcoma, and showed clear immune imaging, indicating that it is hopeful to achieve non-invasive screening of Osteosarcoma patients with PD-L1 positive expression in clinical practice, and this technology might benefit the majority of Osteosarcoma patients.