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Immunotherapy has gained great attention in recent years for its superior antitumor efficacy especially for late-stage tumors, however it suffers from low immunotherapeutic response rate. It is reported that the expression level of PD-L1 is correlated with immunotherapeutic response. The traditional biopsy is invasive and most often randomly sampled, and they cannot accurately reflect the tumor heterogeneity and temporal dynamics in clinics. However, molecular imaging provides chances for noninvasive, dynamic and whole tumor imaging, which can provide the molecular characterization of PD-L1 expression. In this study, anti-PD-L1 antibody was conjugated to the iron oxide nanoparticles to make the PD-L1 targeted imaging probe, abbreviated as aPD-L1@SPIO, which was utilized in combination with magnetic particle imaging (MPI) to image and quantify the PD-L1 expression. The IgG antibody conjugated SPIO was utilized as control probe. The PD-L1 expressing CT26 tumor bearing mice was utilized for MPI, and the imaging probe was administrated through intravenous injection. The MPI imaging was performed at different time points, and the field of view of MPI was chosen below liver. Our targeted PD-L1 MPI imaging data showed that aPD-L1@SPIO, relatively to IgG@SPIO, showed more specific and targeted MPI imaging with longer retention, which was consistent with ex vivo MPI imaging, prussian blue staining and PD-L1 immunohistochemistry. Our study provides us new technique for the highly sensitive, high depth and quantitative imaging for in vivo characterization of immune checkpoint molecular expression in tumors in addition to PET and optical imaging.