The present study examined the clinical significance of serum neuron-specific enolase (NSE) in patients with diffuse large B-cell lymphoma (DLBCL). Serum NSE values were measured using a electrochemiluminescence assay in 106 patients and tissue NSE expression was examined using immunohistochemistry in 65 patients. Fifty-four percent of patients had a positive expression of serum NSE (> 15.20 ng/mL), and cytoplasmic NSE was pathologically demonstrated in 26/65 cases, which showed a positive correlation with that of serum NSE expression. The serum NSE value was closely correlated with performance status, serum lactate dehydrogenase (LDH) level, International Prognostic Index (IPI) score, and Ann Arbor stage, and declined significantly in patients who responded to treatment. In the rituximab-immunochemotherapy group, there was a significant difference in the 5-year overall survival (OS) rate between the NSE-positive and -negative groups (93% vs. 44%, p = 0.001), and the serum NSE level was found to be an independent prognostic factor. Serum NSE may be a novel marker of disease aggressiveness as well as a prognostic factor for DLBCL in the era of rituximab.