Immunotherapy blocking programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) pathway has achieved great therapeutic effect in the clinic, but the overall response rate is not satisfactory. Early studies showed that response to treatment and overall survival could be positively related to PD-L1 expression in tumors. Therefore, accurate measurement of PD-L1 expression will help to screen cancer patients and improve the overall response rate. A small molecular positron emission tomography (PET) probe [¹⁸F]LP-F containing a biphenyl moiety was designed and synthesized for measurement of PD-L1 expression in tumors. The PET probe [¹⁸F]LP-F was obtained with a radiochemical yield of 12.72 ± 1.98%, a radiochemical purity of above 98% and molar activity of 18.8 GBq/μmol. [¹⁸F]LP-F had good stability in phosphate buffer saline (PBS) and mouse serum. In vitro assay indicated that [¹⁸F]LP-F showed moderate affinity to PD-L1. Micro-PET results showed that the tumor accumulation of [¹⁸F]LP-F in A375 tumor was inferior to that in A375-hPD-L1 tumor. All the results demonstrated that [¹⁸F]LP-F could specifically bind to PD-L1 and had a potential application in non-invasive evaluation of PD-L1 expression in tumors.