MicroRNAs (miRNAs) are stably expressed in serum, which could serve as great potential prognostic biomarkers in a variety of diseases, including various cancers. We analyzed the miRNA expression profiles to investigate the role of serum miRNA in predicting response to rituximab, cyclophosphamide, Adriamycin, vincristine, and prednisone (R-CHOP) treatment in diffuse large B cell lymphoma (DLBCL) patients. The present study proceeded through three phases. In the discovery phase, real-time polymerase chain reaction (PCR)-based miRNA profiling was used to test the difference in levels of serum miRNAs between 20 patients with complete remission after 6 cycles of R-CHOP treatment and 20 patients with primary refractory disease matched by age, sex, and stage. After the marker selection phase, the selected serum miRNAs were validated in 133 patients using the quantitative reverse transcriptase-PCR assays during the validation phases. Fifteen serum miRNAs were found to be altered more than 10-fold by real-time PCR-based miRNA profiling between the complete remission and primary refractory groups. The levels of five miRNAs (miR-224, miR-455-3p, miR-1236, miR-33a, and miR-520d-3p) were significantly associated with response to R-CHOP treatment in DLBCL patients. The five-miRNA signature was also a significant predictor of response independent from the International Prognostic Index score. The expression levels of these five serum miRNAs may serve as novel prognostic biomarkers to predict the clinical outcome of DLBCL patients treated with R-CHOP regimen.