Immunotherapy　has　revolutionized　cancer　treatment,　but　its　efficacy　is　severely　hindered　by　the　lack　of　effective　predictors.　Herein,　we　developed　a　homogeneous,　low-volume,　efficient,　and　sensitive　exosomal　programmed　death-ligand　1　(PD-L1,　a　type　of　transmembrane　protein)　quantitation　method　for　cancer　diagnosis　and　immunotherapy　response　prediction　(HOLMES-Exo(PD-L1)).　The　method　combines　a　newly　evolved　aptamer　that　efficiently　binds　to　PD-L1　with　less　hindrance　by　antigen　glycosylation　than　antibody,　and　homogeneous　thermophoresis　with　a　rapid　binding　kinetic.　As　a　result,　HOLMES-Exo(PD-L1)　is　higher　in　sensitivity,　more　rapid　in　reaction　time,　and　easier　to　operate　than　existing　enzyme-linked　immunosorbent　assay　(ELISA)-based　methods.　As　a　consequence　of　an　outstanding　improvement　of　sensitivity,　the　level　of　circulating　exosomal　PD-L1　detected　by　HOLMES-Exo(PD-L1)　can　effectively　distinguish　cancer　patients　from　healthy　volunteers,　and　for　the　first　time　was　found　to　correlate　positively　with　the　metastasis　of　adenocarcinoma.　Overall,　HOLMES-Exo(PD-L1)　brings　a　fresh　approach　to　exosomal　PD-L1　quantitation,　offering　unprecedented　potential　for　early　cancer　diagnosis　and　immunotherapy　response　prediction.