Background <p>Breast cancer (BCa) is the leading cause of cancer-related death among women worldwide. MicroRNAs (miRNAs) are promising tools for diagnosis and prognosis. This study investigated the role of serum miRNAs and tumor markers (TMs) in the diagnosis of BCa.</p> Methods <p>Differentially expressed miRNAs were screened from serum samples of BCa patients and healthy individuals via high-throughput sequencing. The expression of hsa-miR-1911-3p, hsa-miR-4694-5p, hsa-miR-548ao-5p, and hsa-miR-4804-3p in 169 BCa patients and 116 healthy controls was detected via qRT-PCR. Serum tumor-associated antigens were detected by chemiluminescence. Logistic regression was subsequently used to develop the miRNA panel I, TM panel II, and (miRNA + TM) panel III models. Receiver operating characteristic (ROC) curve, precision-recall (PR) curve and decision curve analyses (DCA) were performed to assess the accuracy of the three models for BCa diagnosis. Additionally, the relationships between miRNA expression and the clinical characteristics of patients with BCa were assessed.</p> Results <p>Four serum miRNAs (hsa-miR-1911-3p, hsa-miR-548ao-5p, hsa-miR-4694-5p, and hsa-miR-4804-3p) were newly associated with BCa. The miRNA panel I based on hsa-miR-548ao-5p and hsa-miR-4804-3p showed greater diagnostic effectiveness for BCa than TM panel II based on cancer antigen 125 (CA125) and cancer antigen 153 (CA153), with AUC values of 0.816 and 0.777, respectively. (miRNA + TM) panel III had higher diagnostic effectiveness than miRNA panel I, with an AUC value of 0.870. The expression of miR-548ao-5p and miR-4804-3p is closely related to clinical features, such as human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), progesterone receptor (PR), HER2-enriched subtype, stage III/IV, and lymph node-transplanted breast cancer.</p> Conclusion <p>MiR-548ao-5p and miR-4804-3 could serve as potential biomarkers for the diagnosis of BCa.</p>