The present study examines the clinical significance of serum neuron-specific enolase (NSE) in patients with non-germinal center B-cell type (non-GCB) of diffuse large B-cell lymphoma (DLBCL) that received rituximab chemotherapy. Serum NSE values were measured using electrochemiluminescence assay in 53 patients. About 54.7% of the DLBCL patients had positive expression of serum NSE (>15.20 ng/ml), which closely correlated with performance status, serum LDH level, B symptoms, IPI scores, and Ann-Arbor stages (P < 0.05). The mean serum NSE value in patients with non-GCB subtype of DLBCL was significantly higher than that of GCB subtype of DLBCL (P = 0.001), and among patients in non-GCB subtype group, there was significant difference in the 5-year OS rate between NSE-positive group and negative group (28.3% vs. 81.6%, P < 0.001). Furthermore, serum NSE level was found to be an independent prognostic factor in patients with non-GCB subtype. Serum NSE may be a novel marker of disease aggressiveness as well as a prognostic factor for non-GCB subtype of DLBCL in the era of rituximab.