Breast cancer (BC) screening is important for early detection, but conventional tumor markers lack the desired sensitivity. Aberrant microRNA (miRNA) expression plays an important role in tumor formation and development. Thus, serum miRNAs represent potential BC biomarkers. microRNA-181a (miR-181a) is deregulated in many types of human cancer and is a key oncogenic regulator, but the relationship between serum miR-181a and BC diagnosis has not been investigated. This study investigated serum miR-181a levels in BC patients and healthy controls and compared the diagnostic value of serum miR-181a as a BC tumor marker with the conventional tumor markers CA153 and CEA. Serum miR-181a and miR-16 (as a control) were quantified by real-time quantitative RT-PCR in 20 plasma samples. The promising results prompted analysis of 227 additional samples. The levels of CA153 and CEA were measured using electrochemiluminescence assays. Median miR-181a levels were significantly lower in patients with BC compared to healthy controls (P=0.001). ROC analysis demonstrated the sensitivity and specificity of miR-181a for BC diagnosis at 70.7 and 59.9%, respectively, whereas the sensitivities of CA153 and CEA were 10.53 and 9.21%. As a tumor marker, serum miR-181a expressed a higher level of sensitivity [55.28% (68/123)] in the early stage of BC diagnosis (ductal carcinoma in situ, TNM I and II) than the CA153 and CEA markers (8.13 and 7.32%, respectively). There were no significant associations between miR-181a levels and other clinicopathological parameters. These results suggest that serum miR-181a may represent a novel biomarker for primary BC as well as for early stage BC diagnosis. In combination with other markers, serum miR-181a may improve the sensitivity of BC screening.