In recent years, the cargo profiles of extracellular vesicles (EVs), which were inherited from their parent cells, have emerged as a reliable biomarker for liquid biopsy (LB) in disease diagnosis, prognosis, and treatment monitoring. EVs secreted by different cells exhibit distinct characteristics, particularly in terms of disease diagnosis and prediction. However, currently available techniques for the quantitative analysis of EV cargoes, including enzyme-linked immunosorbent assay (ELISA), cannot specifically identify the cellular origin of EVs, thus seriously affecting the accuracy of EV-based liquid biopsy. In light of this, we here developed ultrabright fluorescent nanosphere (FNs)-based test strips which have the unique capability to specifically assess the levels of PD-L1-positive EVs (PD-L1+ EVs) derived from both tumor cells and immune cells in bodily fluids. The levels of PD-L1+ EV subpopulations in human saliva were quantified using the ultrabright fluorescent nanosphere-based test strips with more convenience and higher efficiency (detection time <30 min). Results demonstrated that the fluorescence intensity of the test line exhibited a good linear relationship respectively with the PD-L1 levels of tumor cell- (R2 = 0.993) and immune cell-derived EVs (R2 = 0.982) in human saliva. By assessing the levels of PD-L1+ EV subpopulations, our test strips hold immense potential for advancing the application of PD-L1+ EV subpopulation-based predictions in tumor diagnosis and prognosis evaluation. In summary, by integrating the benefits of FNs and lateral flow chromatography, we here provide a strategy to accurately measure the cargo levels of EVs originating from diverse cell sources in bodily fluids.