It has been demonstrated that aberrant expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. Recently, specific miRNAs may serve as potential biomarkers for the diagnosis and prognosis of various types of tumor. MiR-23a is known to play important role in the development of cancers and deregulated in various hematological malignancies. The aim of the present study is to explore miR-23a as potential diagnostic and/or prognostic marker of diffuse large B-cell lymphoma (DLBCL). We compared the expression level of miR-23a in DLBCL patients (n = 104) and reactive lymph nodes as controls (n = 28) from formalin-fixed, paraffin-embedded tissues using quantitative reverse transcription-polymerase chain reaction. The expression level of miR-23a was significantly higher in DLBCL patients than in controls (P = 0.001). No significant association was observed between the miR-23a expression level and clinical features such as age, gender, Ann Arbor stage, performance status, lactate dehydrogenase, extranodal sites and International Prognostic Index score (IPI). Kaplan-Meier analysis showed that higher expression level of miR-23a was significantly associated with a poor overall survival (OS) in DLBCL patients (log-rank test, P = 0.029), and multivariable Cox regression revealed the expression of miR-23a (adjusted P = 0.034) and IPI (adjusted P = 0.021) was independently associated with OS. To our knowledge, we provide here the first evidence that miR-23a may represent a diagnostic and prognostic marker for DLBCL. DLBCL patients with a high expression level of miR-23a had a shorter OS than patients with a lower expression level. Further investigation of the changes may be of prognostic significance in clinical practice.