Interaction of human programmed death factor-1 (hPD-1) of T cells and one of its ligands hPD-L1 which is expressed on cancer cells suppresses effector T cell functions. Studies showed that the hPD-1/hPD-L1 pathway is associated with killing mechanisms of tumor cells evading the immune system. Immunotherapy based on the checkpoint inhibitor on hPD-1 has been an important approach to treat cancer; however, not all cancer cells over-express hPD-L1. Detection of hPD-L1 over-expression in cancer cells may be a key factor for deciding on whether immunotherapy should be conducted. In the present study, we produced recombinant hPD-1 using Escherichia coli, and created a fluorescent probe termed quenched hPD-1 (QPD-1) for the detection of hPD-L1. We found that hPD-1 can quench fluorescence of carboxytetramethylrhodamine labeled on its N-terminal and QPD-1 is a convenient tool to rapidly detect hPD-L1 with a limit of detection of 10 nM and detectable range of 10 nM-1000 nM. QPD-1 may also function as a probe to screen for hPD-L1 over-expressing tumor cells and promote appropriate medical procedure through tumor immunotherapy. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.