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 PD‐L1 (HOLMES‐ExoPD‐L1  ) quantitation method for cancer diagnosis and immunotherapy response prediction. 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 rapid binding kinetic. As a result, HOLMES‐ExoPD‐L1  is higher in sensitivity, more rapid in reaction time, and easier to operate than existing ELISA‐based methods. Due to the outstanding improvement of sensitivity, the level of circulating exosomal PD‐L1 detected by HOLMES‐ExoPD‐L1  can effectively distinguish cancer patients from healthy volunteers, and for the first time was found to positive correlation with the metastasis of adenocarcinoma. Overall, HOLMES‐ExoPD‐L1  brings a fresh approach to exosomal PD‐L1 quantitation, offering unprecedented potential for early cancer diagnosis and immunotherapy response prediction.