Objective to explore the specificity and clinical value of specific serum microRNAs (miRNAs) in the early diagnosis of B-cell non-Hodgkin's lymphoma (B-NHL). Methods A total of 115 patients with B-NHL admitted to our hospital from January 2020 to January 2023 were selected as the B-NHL group, and another 100 healthy individuals who underwent physical examinations during the same period were selected as the control group. The expression levels of specific miRNAs (including miRNA-21 and miRNA-155) in the serum of both groups were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The relationship between the expression levels of specific miRNAs and the clinical pathological characteristics of B-NHL patients was analyzed. The diagnostic efficacy of specific miRNAs for B-NHL was analyzed by receiver operating characteristic curve (ROC). Results The relative expression levels of miRNA-21 and miRNA-155 in the serum of the B-NHL group were both higher than those of the control group (P<0.05). There are significant differences in the relative expression levels of miRNA-21 and miRNA-155 in the serum of B-NHL group patients with different Ann Arbor stages, B symptoms, International Prognostic Index (IPI) scores, β2-microglobulin (β2-MG) levels and lactate dehydrogenase (LDH) levels (P<0.05). The results of the ROC curve showed that the AUCs of serum miRNA-21 and miRNA-155 alone in the diagnosis of B-NHL were 0.796 and 0.842, respectively, the sensitivities were 73.30% and 78.29%, respectively, and the specificities were 78.39% and 85.01%, respectively. The positive predictive values were 80.19% and 86.42% respectively, and the negative predictive values were 72.23% and 75.90% respectively. The AUC of the combined detection for diagnosing B-NHL was 0.927, which was higher than that of the single detection, with a sensitivity of 88.35%, a specificity of 90.01%, a positive predictive value of 92.22%, and a negative predictive value of 85.73%. Conclusion serum miRNA-21 and miRNA-155 are highly expressed in patients with B-NHL and are closely related to clinical pathological features. The combined detection can improve the early diagnostic efficiency of B-NHL and can also be used to evaluate the prognosis of patients.