Plasma extracellular vesicles (EVs) have been reported to be a promising source of diagnostic and prognostic biomarkers in various cancers. However, further research in this area is needed due to the limitations of circulating extracellular vesicles detection methods. Using the Single Molecule array (SiMoa) technology, we developed two extracellular vesicle detection assays, CD9-CD63 and PD-L1-CD63, to determine circulating universal EVs and PD-L1 positive EVs, respectively. A total of 164 diffuse large B-cell lymphoma (DLBCL) patients were retrospectively included in this study. Compared with healthy volunteers (n = 25), elevated CD9-CD63 and PD-L1-CD63 signals were detected in the plasma of DLBCL patients (n = 164). High CD9-CD63 signals was associated with molecular subtype, extranodal site and treatment response in DLBCL. A high PD-L1-CD63 signal was also associated with certain clinical features, including extranodal site and treatment response. CD9-CD63 and PD-L1-CD63 signals were found to be important prognostic factors for both progression-free and overall survival. Furthermore, PD-L1-positive EVs were found in all patients, though PD-L1 protein expression was positive in only 35.4% (17/48) of tumor biopsies. No correlation was found between circulating PD-L1+ EVs and soluble PD-L1 (sPD-L1) levels. Our results show that plasma universal EV and PD-L1-positive EV levels are significantly elevated in DLBCL and might serve as biomarkers for predicting survival outcomes in DLBCL patients.Keywords: DLBCL; PD-L1; extracellular vesicles; lymphoma; plasma.