Background <p>This study investigates the differential expression of ENO2 in serum extracellular vesicles (EVs) of patients with diffuse large B cell lymphoma (DLBCL) and its correlation with clinicopathological characteristics and prognosis.</p> Methods <p>Serum samples before receiving treatment, clinicopathological characteristics, and follow-up information were collected from 78 patients with DLBCL, and 40 patients with reactive hyperplasia of lymph nodes (RH) were selected as the control group. ENO2 expression in serum EVs was detected by RT-qPCR and Western blot. The diagnostic value of ENO2 in serum EVs for DLBCL was analyzed by ROC curve. The correlation between ENO2 expression and clinicopathological data was analyzed by chi square test. The 5-year overall survival (OS) was analyzed by Kaplan–Meier method. The COX proportional hazards model was employed for analyzing prognostic risk factors of DLBCL.</p> Results <p>ENO2 is highly expressed in serum EVs of DLBCL patients. ENO2 in serum EVs has moderate diagnostic efficacy for DLBCL, with a sensitivity, specificity, and AUC of 0.808, 0.725, and 0.789, respectively. ENO2 expression is closely related to ECOG score, LDH level, Ann Arbor stage, IPI, and Bcl-2. DLBCL patients with age (&gt; 60&#xa0;years), ECOG (2–4), LDH level (High), Ann Arbor stage (III + IV), IPI score (3–5), Ki-67 (≥ 70%), Bcl-2 (positive), and ENO2 protein (high expression) have lower 5-year OS. ENO2 protein (high expression), ECOG (2–4), Ann Arbor stage (III + IV), and Ki-67 (≥ 70%) are independent prognostic risk factor for DLBCL.</p> Conclusions <p>ENO2 is correlated with the clinicopathological characteristics of DLBCL and is an independent prognostic risk factor.</p>